CN103841976A - Combinations of AKT AND MEK inhibitor compounds, and methods of use - Google Patents

Abstract

The invention provides combinations comprising a) compound of formula I: (formula I), or a pharmaceutically acceptable salt thereof; and another agent selected from GDC-0973, PD-0325901, or a pharmaceutically acceptable salt thereof. The combinations are particularly useful for treating hyperproliferative disorders, such as cancer.

Description

The combination of AKT and mek inhibitor compound and using method thereof

The cross reference of related application

The application requires the priority of the U.S. Provisional Application 61/471,038 of submitting on April 1st, 2011.The full content of this provisional application mode is by reference incorporated to the application.

Technical field

The present invention relates generally to the drug regimen of the active compound with anti-excess proliferative disease such as cancer and it comprises the compound that suppresses AKT kinase activity.The invention still further relates to the method for described combination for external, original position and in-vivo diagnostic or treatment mammalian cell or related diseases disease of science that use.

Background technology

Protein kinase (PK) is by shifting end (γ) phosphate ester on ATP, the enzyme of the phosphorylation of hydroxyl in tyrosine, serine and the threonine residues of catalytic protein.Pass through signal transduction pathway, these enzymes regulate Growth of Cells, Differentiation and proliferation, the nearly all aspect that is cell cycle all depends on PK activity (Hardie, and Hanks G., S. (1995) The Protein Kinase Facts Book.I and II, Academic Press, San Diego, CA).In addition, abnormal PK activity has related to many diseases, and scope is from for example extremely extremely fatal for example glioblastoma of disease (brain cancer) of psoriasis of relatively non-life-threatening disease.Protein kinase is the important targeting classification (Cohen, P. (2002) Nature Rev.Drug Discovery1:309) that treatment regulates.

International Patent Application Publication WO2008/006040 has discussed numerous AKT inhibitor, comprise compound (S)-2-(4-chlorphenyl)-1-(4-((5R, 7R)-7-hydroxy-5-methyl base-6,7-dihydro-5H-cyclopenta [d] pyrimidine-4-yl) piperazine-1-yl)-3-(isopropylamino) third-1-ketone (formula I):

At present, still exist for the demand of method and composition of improvement that can be used for overmedication proliferative disease such as cancer.

Summary of the invention

Additive effect or the cooperative effect of having determined the growth of in vitro and in vivo anticancer can realize by the combination of the compound of giving construction I or its pharmaceutical salts and some other certain drug.Described combination and method can be used for overmedication proliferative disease such as cancer.

Therefore, certain embodiments of the present invention provide the method that is used for the treatment of excess proliferative disease in mammal, comprise to described mammal and combining, describedly be combined as compound or its pharmaceutical salts of formula I and be selected from the combination of the other medicine of GDC-0973, PD-0325901 or its pharmaceutical salts, the compound of described formula I is:

In certain embodiments, described excess proliferative disease is cancer.

In certain embodiments, described cancer is relevant to PTEN sudden change.

In certain embodiments, described cancer is suddenlyd change to AKT, is crossed and express or increase relevant.

In certain embodiments, described cancer is relevant to PI3K sudden change.

In certain embodiments, described cancer is relevant to Her2/ErbB2 amplification.

In certain embodiments, described cancer is selected from mesothelioma, carcinoma of endometrium, cancer of pancreas, breast carcinoma, pulmonary carcinoma, ovarian cancer, carcinoma of prostate, melanoma, gastric cancer, colon cancer, renal carcinoma, head and neck cancer and glioma.

In certain embodiments, the combination of the compound of giving construction I or its pharmaceutical salts and GDC-0973 or its pharmaceutical salts.

In certain embodiments, the combination of the compound of giving construction I or its pharmaceutical salts and PD-0325901 or its pharmaceutical salts.

In certain embodiments, the compound of giving construction I or the combination of its salt and described one or more medicines simultaneously.

In certain embodiments, the successively compound of giving construction I or its salt and described one or more medicines.

In certain embodiments, within approximately 1 to approximately 10 day before giving described combination, start to give described one or more medicines.

In certain embodiments, before giving described combination, within approximately 1 to approximately 10 day, start compound or its salt of giving construction I.

In certain embodiments, starting on the same day compound or its salt of giving construction I and giving described one or more medicines.

Certain embodiments of the present invention provide compound or its pharmaceutical salts of formula I, and it for improving the therapeutic use for the patient's of excess proliferative disease treatment quality of life together with being selected from the medicine of GDC-0973 and PD-0325901.

Certain embodiments of the present invention provide and have been used for the treatment of the disease that regulated by AKT kinases in mammal or the method for disease, comprise to described mammal and give a) compound or its pharmaceutical salts of formula I; And b) be selected from one or more medicines of GDC-0973 and PD-0325901.

Certain embodiments of the present invention provide a) compound or its pharmaceutical salts of formula I; And b) being selected from the combination of one or more medicines of GDC-0973 and PD-0325901, it is used for the treatment of excess proliferative disease.

Certain embodiments of the present invention provide a) compound or its pharmaceutical salts of formula I; And b) being selected from the combination of one or more medicines of GDC-0973 and PD-0325901, it is used for the treatment of the disease or the disease that are regulated by AKT kinases.

Certain embodiments of the present invention provide the compound of formula I or being combined in for the preparation of the purposes in the medicine of excess proliferative disease in treatment mammal of its pharmaceutical salts and GDC-0973 and PD-0325901.

Certain embodiments of the present invention provide the compound of formula I or being combined in for the preparation of the purposes in the medicine of the disease being regulated by AKT kinases in treatment mammal or disease of its pharmaceutical salts and GDC-0973 and PD-0325901.

Certain embodiments of the present invention provide the test kit that is used for the treatment of excess proliferative disease, package insert or label that it comprises compound or its pharmaceutical salts, the container of formula I and indicates the compound of giving construction I and be selected from one or more medicines of GDC-0973 and PD-0325901.

Certain embodiments of the present invention provide product, comprise and have compound or its pharmaceutical salts of formula I and be selected from GDC-0973 and one or more medicines of PD-0325901; Described product is as the compound artifact for separately, simultaneously or successively using at overmedication proliferative disease.

In the time that many cell types comprise the combination that gives GDC-0068 and GDC-0973 in melanoma, pulmonary carcinoma, colon cancer, ovarian cancer, renal carcinoma, breast carcinoma, carcinoma of prostate, pancreatic carcinoma, observe collaborative/additive effect when external, and these discoveries are confirmed in melanoma, colon cancer and pulmonary carcinoma xenograft models in vivo.In the tumor type being driven by Ras/Raf or two kinds of pathway activations, observe cooperative effect.When give the combination of GDC-0068 and GDC-0973 in various kinds of cell time, for example, in melanoma, pulmonary carcinoma (NSCLC) and colon carcinoma cell line show synergistic.In the time giving the combination of GDC-0068 and GDC-0973, breast cancer cell (comprising lumen (ER+), Her2+ and substrate triploid negative breast cancer (basal triple negative breast cancers)) also can show synergistic.In the time giving the combination of GDC-0068 and GDC-0973, even also observe cooperative effect in the cell of the Meki sensitivity to independent.

The mutation status that has been found that cancerous cell is the biomarker how described cancerous cell responds different therapeutic schemes.For example, the cancerous cell that has a combination of (for example PI3K or the AKT) sudden change of PI3K approach and Kras and/or Braf sudden change can show the positive (for example cooperative effect) response to the combined therapy described in the application.In addition, the PTEN state of cancerous cell is also biomarker.Therefore, certain embodiments of the present invention comprise the method for the treatment of the cancerous cell (external or body in) of the combination with these biomarkers with these combination treatments.Certain embodiments of the present invention comprise the patient for combined therapy who selects the combination with these biomarkers.

In A2058 (PTEN without/Braf mutant) melanoma model, observe the strong cooperative effect of GDC-0068 and GDC-0973 combination.Observe suitable single medicine tumor growth in the higher dosage of all dosage and the GDC-0068 of GDC-0973 (75 and 100mg/kg) and suppress (TGI).GDC-0068 at 50mg/kg does not observe TGI.Being combined in this model of two kinds of medicines is well tolerable, and wherein maximum weight loss is for～13%.

Except providing for given excess proliferative disease the treatment of improvement, the quality of life that gives to improve patient of some combination of the present invention (quality of life of experiencing than the same patient of accepting different treatments).For example, the quality of life (by the quality of life of experience, condition is that described patient only accepts the chemotherapeutant as treatment than same patient) of improvement can be provided to the combination of the medicine described in compound or its pharmaceutical salts and the application of patient's giving construction I.For example, use the combination treatment of the combination described in the application can reduce the dosage of required therapeutic agent, weaken thus the side effect relevant to high dose chemotherapeutant (for example feel sick, vomiting, alopecia, erythra, appetite decline, lose weight etc.).Described combination also can cause the tumor load of reduction and relevant adverse events, such as pain, organ dysfunction, lose weight etc.Therefore, one aspect of the present invention provides compound or its pharmaceutical salts of formula I, its for the therapeutic use improving together with medicine described in the application for the patient's of excess proliferative disease treatment quality of life.

Accompanying drawing explanation

The result of the combination (2.5mg/kg) that Fig. 1 example has illustrated GDC-0068 and GDC-0973 to gross tumor volume.

The result of the combination (5.0mg/kg) that Fig. 2 example has illustrated GDC-0068 and GDC-0973 to gross tumor volume.

The result of the combination (7.5mg/kg) that Fig. 3 example has illustrated GDC-0068 and GDC-0973 to gross tumor volume.

Fig. 4 example has illustrated the result that the combination of GDC-0068 and GDC-0973 to external colorectal cancer cell is.

The result of the combination that Fig. 5 example has illustrated GDC-0068 and GDC-0973 to HCT-116 (colon cancer-PI3K and Kras mutant).Shown two dimension (2D) thermal map (heatmap), it has shown the combined effect of the cell survival to HCT-116 cell.Show and increased the GDC-0068 of concentration and shown the GDC-0973 that increases concentration at y-axle at x axle.Shown inhibition percent (% inhibition) thermal map on right side, it indicates in combination or as the GDC-0068 of each concentration and the inhibition percent of GDC-0973 of single medicine; The contrast that is exposed to vehicle (DMSO) is set to 0.Mark to calculating BLISS with thermal map for the each dosage showing in left side.

The result of the combination that Fig. 6 example has illustrated GDC-0068 and GDC-0973 to external NSCLC cell line.

The result of the combination that Fig. 7 example has illustrated GDC-0068 and GDC-0973 to H2122 (NSCLC-Kras mutant).Two dimension (2D) thermal map demonstrates the combined effect of the cell survival to NCI-H2122 cell.Show and increased the GDC-0068 of concentration and shown the GDC-0973 that increases concentration at y-axle at x-axle.Shown inhibition percent (% inhibition) thermal map on right side, it indicates in combination or as the GDC-0068 of each concentration and the inhibition percent of GDC-0973 of single medicine; The contrast that is exposed to vehicle (DMSO) is set to 0.Mark to calculating BLISS with thermal map for the each dosage showing in left side.

Fig. 8 example has illustrated that the combination of GDC-0068 and GDC-0973 is to the result of external K-1735.

The result of the combination that Fig. 9 example has illustrated single medicine and GDC-0068 and GDC-0973 to A2058 (melanoma-PTEN-/-and Braf mutant).Shown two dimension (2D) thermal map, it demonstrates the combined effect of the cell survival to A2058 cell.Mark to calculating BLISS with thermal map for the each dosage showing in left side.Show and increased the GDC-0068 of concentration and shown the GDC-0973 that increases concentration at y-axle at x-axle.Shown inhibition percent (% inhibition) thermal map on right side, it indicates in combination or as the GDC-0068 of each concentration and the inhibition percent of GDC-0973 of single medicine; The contrast that is exposed to vehicle (DMSO) is set to 0.

Figure 10 example has illustrated knocks down effect (knockdown) than the enhancing of the AKT of single medicine and MEK pathway activities.

The result of the combination that Figure 11 example has illustrated GDC-0973 and GDC-0068 to MDA-MB-468 breast cancer cell line.

Figure 12 example has illustrated that the combination of GDC-0068 and GDC-0973 is to the result of external breast cancer cell line.

Figure 13 example has illustrated that the combination of GDC-0068 and GDC-0973 is to the result of ovarian cancer.

Figure 14 example has illustrated that the combination of GDC-0068 and GDC-0973 is to the result of external prostate cancer cell line.

Figure 15 example illustrated oral administration GDC-0068+GDC-0973 (mek inhibitor) be combined in the result in MX-1 breast tumor.

Figure 16 example illustrated oral administration GDC-0068+GDC-0973 (mek inhibitor) be combined in the result in H2122NSCLC tumor.

Figure 17 example illustrated oral administration GDC-0068+GDC-0973 (mek inhibitor) be combined in the result in SW1990 pancreas tumor.

Figure 18 example illustrated oral administration GDC-0068+GDC-0973 (mek inhibitor) be combined in the result in Pa_Tu-8902 pancreas tumor.

Figure 19 example illustrated oral administration GDC-0068+GDC-0973 (mek inhibitor) be combined in the result in 537Mel melanoma.

Figure 20 example illustrated oral administration GDC-0068+GDC-0973 (mek inhibitor) be combined in the result in A2058 melanoma.

Figure 21 example illustrated oral administration GDC-0068+GDC-0973 (mek inhibitor) be combined in the result in HCT-116 colorectum tumor.

Figure 22 a-22b has shown the result that compares the inhibition of single medicine and the cell survival of combination treatment to various cell lines.GDC-0068 cell usefulness is associated with the Akt activation being caused by the change of PI3K/PTEN/HER2, and GDC-0973 cell usefulness is associated with the MEK activation causing that suddenlyd change by RAS or B-RAF.GDC-0068-and GDC-0973-sensitivity cell system are normally mutually exclusive.The cell line of approximately 1/3rd test demonstrates the resistance (referring to Figure 22 a-22b) to two kinds of medicines.In the cell line of great majority test, than the single medicine of independent use, the combination of GDC-0068 and GDC-0973 causes the inhibition of the enhancing to cell survival.Use BLISS dependent/non-dependent model evaluation combined effect (Leh á r et al.2007).

On Figure 22 a, illustrated example has illustrated the single medicine IC50 for GDC-0068 and GDC-0973 in multiple cancerous cell line.By cell with increasing the GDC-0068 of concentration or GDC-0973 or using CelTiter-Glo to measure with the combined treatment of RPMI+10%FBS and after 4 days for survival rate.Corresponding lower illustrated example has illustrated for the GDC-0068 of some specific gene types and the cooperative effect of GDC-0973 combination.Painted module represents sudden change, disappearance or activation.Sudden change/change of B-RAF, RAS, HER2, PI3K or PTEN under every kind of cell line by painted square represent (B-RAF, brown; RAS, redness; HER2, blueness; PTEN, bottle green; For PI3K, light green color represents the kinases territory sudden change of PIK3CA, sudden change or the amplification in light blue expression nonkinase territory).PTEN changes signal or the gene mutation that expression can not be detected by western blot for this albumen.Also represent by different colors and letter for the tissue-derived of every kind of cell line, i.e. breast carcinoma (Br), colon cancer (Co), nonsmall-cell lung cancer (Lu), melanoma (Me), ovarian cancer (Ov), carcinoma of prostate (Pr) and renal carcinoma (Re).

Figure 22 b example has illustrated the overall positive combination Bliss scoring in cell multiplex system for GDC-0068 and GDC-0973.In cell multiplex system, observe cooperative effect, it represents by overall positive BLISS scoring, particularly there is the cell line of RAS/RAF pathway activation or there is PI3K/Akt and the cell line of RAS/RAF pathway activation in.

Overall positive BLISS scoring is by the combination calculation of the GDC-0068 in every kind of cell line and GDC-0973.

Figure 23 example has illustrated that Bliss thermal map and the % in 537MEL melanoma (PTEN without, Brafamp/del) suppresses for GDC-0068 and GDC-0973; The combination of GDC-0068 and GDC-0973 suppresses two kinds of approach and increases cell death.

Figure 24 example has illustrated for the protein mottling analysis of processing the people HTC116 colon carcinoma cell line of 24 hours by GDC-0068 and GDC-0973 cell line.The GDC-0068 of HCT-116 cell and certain concentration and GDC-0973 are cultivated approximately 3 hours.The phosphorylation of Akt, MEK and their downstream labelling is analyzed by western blot.

Figure 25 example has illustrated that GDC-0068 and GDC-0973 combination has increased the effect in 537MEL melanoma (PTEN without, Brafamp/del).

Figure 26 example illustrated with respect to vehicle in contrast, for the significant change of the phosphoprotein expression of GDC-0068 and GDC-0973 combination.Mice is being given to GDC-0068 (100mg/kg) or the GDC-0973 (7.5mg/kg) of single dose or combining and within latter 3 hours, collect A2058x1 tumor.Use anti-phase protein arrays (RPPA) to analyze tumor.

Figure 27 example illustrated in A2058 xenograft tumor after administration, for single medicine, for the significant change of the phosphoprotein expression of GDC-0068 and GDC-0973 combination.

Figure 28 example has illustrated two dimension (2D) thermal map, and it demonstrates the combined effect of the cell survival to MALME3M cell.Show and increased the GDC-0068 of concentration and shown the GDC-0973 that increases concentration at y-axle at x-axle.Suppress that percent (% inhibition) thermal map demonstrates in combination or as the GDC-0068 of each concentration and the inhibition percent of GDC-0973 of single medicine; The contrast that is exposed to vehicle (DMSO) is set to 0.

Figure 29 example has illustrated two dimension (2D) thermal map, and it demonstrates the combined effect of the cell survival to MALME3 cell.Show and increased the GDC-0068 of concentration and shown the GDC-0973 that increases concentration at y-axle at x-axle.Suppress that percent (% inhibition) thermal map demonstrates in combination or as the GDC-0068 of each concentration and the inhibition percent of GDC-0973 of single medicine; The contrast that is exposed to vehicle (DMSO) is set to 0.Mark to calculating BLISS with thermal map for the each dosage showing on right side.

Figure 30 example has illustrated two dimension (2D) thermal map, and it demonstrates the combined effect of the cell survival to NCI-BL2122 cell.Show and increased the GDC-0068 of concentration and shown the GDC-0973 that increases concentration at y-axle at x-axle.Suppress that percent (% inhibition) thermal map demonstrates in combination or as the GDC-0068 of each concentration and the inhibition percent of GDC-0973 of single medicine; The contrast that is exposed to vehicle (DMSO) is set to 0.Mark to calculating BLISS with thermal map for the each dosage showing on right side.

Figure 31 has shown the variation (24 hours) of phosphoprotein expression and has used the combination of GDC-0068 and GDC-0973 to regulate AKT and MEK approach.Mice is being given to GDC-0068 (100mg/kg) or the GDC-0973 (7.5mg/kg) of single dose or combining and within latter 24 hours, collect A2058x1 tumor.Use anti-phase protein arrays (RPPA) to analyze tumor.

The specific embodiment

The word using in this description and claim " comprises/comprises (comprise, comprising, include, including and includes) " and be intended to illustrate the existence of described feature, entirety, component or step, but they are not got rid of and exist or add one or more further features, entirety, component, step or their combination.

Term " treatment (treat) " and " treatment (treatment) " refer to that therapeutic disposes and preventive measure, wherein object be prevention slow down (alleviating) less desirable physiology's variation or obstacle as the growth of cancer, development or diffusion.For purposes of the present invention, clinical effectiveness useful or that expect includes but not limited to relief of symptoms, reduces lesion degree, stablizes (not being to worsen) morbid state, postpones or slows down progression of disease, improvement or relaxes morbid state and improvement (part takes a turn for the better or improvement completely), and no matter these results are detectable or undetectable." treatment " also can represent the survival of prolongation compared with the expection survival of not receiving treatment.Need the object for the treatment of to comprise the object that the object suffering from the object of disease or obstacle and easily suffer from described disease or obstacle or described disease or obstacle should be prevented.

Phrase " treatment effective dose " represents the amount of the compounds of this invention of disease specific, disease or the obstacle of (i) treatment or prevention the application description, (ii) weaken, improve or eliminate the amount of the compounds of this invention of one or more symptoms of disease specific, disease or obstacle that the application describes, or (iii) prevention or postpone the amount of the compounds of this invention of the outbreak of one or more symptoms of disease specific, disease or obstacle that the application describes.In the situation of cancer, the medicine for the treatment of effective dose can reduce the quantity of cancerous cell; Reduce tumor size; Suppressing (slow down to a certain extent and preferably stop) cancerous cell infiltrates in peripheral organs; Suppress to a certain extent tumor growth; And/or alleviate to a certain extent one or more symptoms relevant to cancer.If medicine can prevent the growth of cancerous cell and/or kill existing cancerous cell, it may be (cytostatic) of cell growth inhibition and/or Cytotoxic.For treatment of cancer, can for example measure effect by evaluating progression of disease time (TTP) and/or definite response rate (RR).

Term " cancer (cancer) " and " (cancerous) of cancer " refer to or describe feature in mammal and be typically the physiological conditions of unadjusted Growth of Cells." tumor " comprises one or more cancerous cell.The example of cancer includes but not limited to carcinoma (carcinoma), lymphoma, blastoma, sarcoma and leukemia or lymph sample malignant tumor.The example more specifically of described cancer comprises squamous cell carcinoma (for example epithelium squamous cell carcinoma), pulmonary carcinoma comprises small cell lung cancer, nonsmall-cell lung cancer (" NSCLC "), adenocarcinoma of lung (adenocarcinoma of the lung) and squamous cell lung carcinoma (squamous carcinoma of the lung), peritoneal cancer, hepatocarcinoma, gastric cancer (gastric or stomach cancer) comprises human primary gastrointestinal cancers, cancer of pancreas, glioblastoma, cervical cancer, ovarian cancer, hepatocarcinoma (liver cancer), bladder cancer, hepatoma (hepatoma), breast carcinoma (breast cancer), colon cancer, rectal cancer, colorectal carcinoma, carcinoma of endometrium or uterus carcinoma, salivary-gland carcinoma, renal carcinoma or renal cancer, carcinoma of prostate, carcinoma vulvae (vulval cancer), thyroid carcinoma, liver cancer (hepatic carcinoma), anus cancer, carcinoma of penis, and head and neck cancer.Gastric cancer used in this application comprises gastric cancer (stomach cancer), and it can and can spread in any part development of stomach and spread all over stomach everywhere and diffuse to other organ; Particularly esophagus, lung, lymph node regulating liver-QI.

" chemotherapeutant " is biology (macromole) or chemistry (micromolecule) compound that is used for the treatment of cancer (irrelevant with mechanism of action).

" platinum agent " is the chemotherapeutant that comprises platinum, for example carboplatin, cisplatin and oxaliplatin.

Term " mammal " includes but not limited to people, mice, rat, Cavia porcellus, monkey, Canis familiaris L., cat, horse, cattle, pig, sheep and domestic animal.Term patient refers to mammal, and in one embodiment, described patient behaves.

The term " package insert (package insert) " using refers to the description in the commercially available back that is usually included in treatment product, it contains the information about indication, usage, dosage, administration, contraindication and/or warning item, and these information relate to the use of above-mentioned treatment product.

Phrase used in this application " pharmaceutical salts " refers to the medicinal organic-or-inorganic salt of the compounds of this invention.Exemplary salt includes but not limited to sulfate, citrate, acetate, oxalates, chloride, bromide, iodide, nitrate, disulfate, phosphate, acid phosphate, .gamma.-pyridinecarboxylic acid salt, lactate, Salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, biatrate, Ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formates, benzoate, glutamate, Glu, mesylate (methanesulfonate) " mesylate (mesylate) ", esilate, benzene sulfonate, tosilate and embonate are (, 1, 1'-methylene-bis-(2-hydroxyl-3-naphthoate)).Pharmaceutical salts can relate to and comprise another molecule, such as acetate ion, succinate ion or other counter ion counterionsl gegenions.Counter ion counterionsl gegenions can be any organic or inorganic part (moiety) of the electric charge on stable matrix compound.And pharmaceutical salts can have and exceedes a charge atom in its structure.The situation of multiple charge atoms is that the part of pharmaceutical salts can have multiple counter ion counterionsl gegenions.Therefore, pharmaceutical salts can have one or more charge atoms and/or one or more counter ion counterionsl gegenions.

Expection pharmaceutical salts can by this area can with any proper method prepare.For example, by mineral acid or organic acid processing for free alkali, the all example hydrochloric acids of described mineral acid, hydrobromic acid, sulphuric acid, nitric acid, methanesulfonic acid, phosphoric acid etc., described organic acid such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, acetone acid, oxalic acid, glycolic, salicylic acid, pyrans glycosyl acid (pyranosidyl acid) are if glucuronic acid or galacturonic acid, 'alpha '-hydroxy acids are if citric acid or tartaric acid, aminoacid are if aspartic acid or glutamic acid, aromatic acid are if benzoic acid or cinnamic acid, sulfonic acid are as p-methyl benzenesulfonic acid or ethyl sulfonic acid etc.It has been generally acknowledged that the acid that is suitable for being formed by alkaline pharmaceutical compound medicinal or acceptable salt is discussed at for example P.Stahl et al, Camille G. (eds.) Handbook of Pharmaceutical Salts.Properties, Selection and Use. (2002) Zurich:Wiley-VCH; S.Berge et al, Journal of Pharmaceutical Sciences (1977) 66 (1) 119; P.Gould, International J.of Pharmaceutics (1986) 33201217; Anderson et al, The Practice of Medicinal Chemistry (1996), Academic Press, New York; Remington ' sPharmaceutical Sciences, 18 ^thed., (1995) Mack Publishing Co., Easton PA; With The Orange Book (Food & Drug Administration, Washington, D.C.on their website).These open modes are by reference incorporated to the application.

Phrase " medicinal " represents that described material or compositions must comprise that with other composition preparation and/or mammal to be treated adapt chemically and/or in toxicology.

" cooperative effect " used in this application refers to the more effective therapeutic combination of additive effect than two or more single medicine.Concertedness between a kind of in the compound of formula I or its pharmaceutical salts and GDC-0973 and PD-0325901 interacts and can determine according to the result being obtained by the mensuration described in the application.The result of these mensuration can be analyzed by Chou and Talalay combined method and with the docs-effect analysis of CalcuSyn software, to obtain combinatorial index (Chou and Talalay, 1984, Adv.Enzyme Regul.22:27-55).In some mensuration systems, evaluated the combination that the application provides, and described data can adopt for the standardization program of the cooperative effect between quantitative anticarcinogen, additive effect and antagonism and analyze.Illustrative examples is described in Chou and Talalay, in " New Avenues in Developmental Cancer Chemotherapy, " Academic Press, 1987, Chapter2.Combinatorial index is less than 0.8 expression cooperative effect, and described value is greater than 1.2 expression antagonisms and described value represents additive effect between 0.8 to 1.2.Described combination treatment can provide " concertedness " and confirm " cooperative effect ", and the effect realizing when described active component uses is together greater than the summation by the effect that separately uses described compound to obtain.Cooperative effect can obtain under the following conditions: (1) in the time jointly preparing described active component and give simultaneously or send in the unit dose formulations of combination; (2) when alternately sending described active component as the preparation separating or when parallel sending; Or (3) when by certain other scheme.In the time sending with rotational therapy, cooperative effect can be come to obtain when successively giving or send described compound for example carrying out different injections by the syringe separating.Generally speaking,, in rotational therapy process, successively give continuously every kind of active component of effective dose, and in combination treatment, give together two kinds or more kinds of active component of effective dose.Use BLISS dependent/non-dependent model and the highest single medicine (HSA) model to evaluate combined effect (Leh á r et al.2007, Molecular Systems Biology3:80).BLISS scoring is carried out quantitative and positive BLISS scoring (being greater than 0) to the potentiation degree of single medicine and is shown to be greater than simple cumulative.The positive BLISS scoring of accumulation is greater than 250 and is considered to be in viewed strong cooperative effect in tested concentration range.HAS scoring (being greater than 0) shows that combined effect is greater than at the maximum single medicine of respective concentration and replys.

An aspect is included in the method for carrying out tumor growth inhibition (TGI) in the patient who suffers from the cancer that comprises PI3K, AKT or PTEN sudden change, and described cancer further comprises in an example that RAS/RAF sudden change, described method comprise to described patient and gives or its pharmaceutical salts in GDC-0068 and GDC-0973 and PD-0325901.In certain embodiments, described in, be combined as synergitic.In certain embodiments, the TGI of described combination is greater than the independent TGI of in GDC-0068 or GDC-0973 and PD-0325901.In certain embodiments, the TGI of described combination is greater than approximately 10,15,20,25,30,35,40,45,50,55,60,65,70 or 75% of the independent TGI of in GDC-0068 or GDC-0973 and PD-0325901.

The method of measuring TGI is known in the art.In an illustrative methods, determined mean tumour volume and before treatment and treatment after patient between compare.Gross tumor volume can two dimension (length and width) measure, its use this area for example UltraCal IV caliper of any method (Fred V.Fowler Company) or through PET (positron emission tomography) or some other method.Can use formula: gross tumor volume (mm ³)=(length x width ²) x0.5.Lasting multiple time periods measurement gross tumor volumes can use linear hybrid effect (LME) method (Pinheiro et al.2009) of hybrid modeling to complete.The method can solve repeated measure (and multiple patient).Cubic regression batten can be used for nonlinear Distribution to fit to the time-histories at the gross tumor volume of each dosage level.Then these nonlinear Distribution can be associated with the dosage in mixed model.Suppress to can be used as the calculating of getting off as the tumor growth of vehicle percent: area under the matched curve of the every day relevant to vehicle (AUC) percent, it uses following formula:

Use this formula, 100% TGI value representation tumor is stagnated, and is greater than approximately 1% but be less than approximately 100% TGI value representation tumor growth and suppress, and is greater than approximately 100% TGI value representation tumor regression.

the preparation of the compound of formula I

The compound of formula I and salt thereof can be as described at International Patent Application Publication WO2008/006040 or as preparing described in following embodiment 1.In the compound of preparation formula I, may need the far-end functional group (such as primary amine or secondary amine etc.) to intermediate to protect.To depend on the character of described far-end functional group and the condition of preparation method and change for the demand of described protection.Suitable amino-protecting group (NH-Pg) comprises acetyl group, trifluoroacetyl group, tert-butoxycarbonyl (BOC), benzyl oxygen base carbonyl (CBz) and 9-fluorenyl methylene oxygen base carbonyl (Fmoc).Demand for described protection can easily be determined by those skilled in the art.Generality for blocking group and their purposes is described, referring to T.W.Greene, and Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

separation method

In any synthetic method of preparation I compound, by disconnected from each other product and/or from initiation material separate be favourable.Utilize ordinary skill, by the required product separation of each step or series of steps and/or be purified to the target uniformity.Typically, this separation comprises heterogeneous extraction, crystallization from solvent or solvent mixture, distillation, distillation or chromatograph.Chromatograph can comprise many methods, for example comprises: anti-phase and normal-phase chromatography; Volume removing chromatogram; Ion exchange chromatography; High, in and low pressure liquid chroma tography and device; Little type analysis; Simulation moving-bed (SMB) and preparative thin layer chromatography or thick layer chromatography method, and small-scale thin layer and flash chromatography technology.

Another kind of separation method comprises: with the agent treated reactant mixture of selecting, with target product, unreacted initiation material, byproduct of reaction etc. in conjunction with or it is separated.This reagent comprises adsorbent or absorbent such as activated carbon, molecular sieve, Ion Exchange Medium etc.Selectively, reagent can be acid (in the situation that of basic matterial), alkali (in the situation that of acidic materials), binding reagents such as antibody, in conjunction with albumen, such as crown ether, liquid/liquid ion extractuin reagent (LIX) etc. of chelating agen optionally.

Select suitable separation method according to the character of related material.For example, boiling point and molecular weight in distillation and distillation, the existence in chromatograph or do not have polar functional group, the material stability in acidity and alkaline medium in heterogeneous extraction etc.Those skilled in the art can apply most suitable technology, to realize required separation.

Based on physical chemistry difference, utilize method well known to those skilled in the art, for example chromatograph and/or fractional crystallization, can be separated into non-enantiomer mixture its single diastereomer.Enantiomer can separate as follows: by with suitable optically active compounds (for example chiral auxiliary, for example chiral alcohol or Mosher's acyl chlorides) reaction, enantiomeric mixture is converted into non-enantiomer mixture, separate diastereomer, and for example, be corresponding pure enantiomer by single diastereomer conversion (hydrolysis).Equally, compounds more of the present invention may be atropisomer (biaryl for example replacing), and think that it is a part of the present invention.Can also use chirality HPLC post to separate enantiomer.

Single stereoisomer, for example enantiomer (not basically containing its stereoisomer) can be used the method that for example uses optical activity resolution reagent to form diastereomer, obtains by splitting racemic mixture.(Eliel,E.and?Wilen,S."Stereochemistry?of?Organic?Compounds,"John?Wiley&Sons,Inc.,New?York,1994;Lochmuller,C.H.,J.Chromatogr.,(1975)113(3):283-302)。The racemic mixture of chipal compounds of the present invention separates by arbitrary suitable method and separates, it comprises: (1) forms ion, diastereomeric salt with chipal compounds, and separate by fractional crystallization or other method, (2) form diastereomeric compound with chiral derivatization reagent, separate diastereomer, and be converted into pure stereoisomer, (3), under chirality condition, directly separate stereoisomer pure or enrichment substantially.Referring to: " Drug Stereochemistry, Analytical Methods and Pharmacology, " Irving W.Wainer, Ed., Marcel Dekker, Inc., New York (1993).In method (1), diastereomeric salt can be formed with the reacting of asymmetric compound of carrying for example carboxylic acid of acidic functionality and sulfonic acid by the chiral base of enantiomeric pure such as brucine, quinine, ephedrine, strychnine, Alpha-Methyl-β-phenethylamine (amfetamine) etc.By fractional crystallization or chromatography of ions, can induce separation diastereomeric salt.For the separation of the optical isomer of amino-compound, add chiral carboxylic acids or sulfonic acid, for example camphorsulfonic acid, tartaric acid, mandelic acid or lactic acid, can cause the formation of diastereomeric salt.

Selectively, by method (2), make the substrate that will split and an enantiomerism precursor reactant of chipal compounds, form diastereomer to (E.and Wilen, S. " Stereochemistry of Organic Compounds ", John Wiley & Sons, Inc., 1994, p.322).Diastereomeric compound can be formed with for example the reacting of menthyl derivatives of chiral derivatization reagent of enantiomeric pure by asymmetric compound, then separates diastereomer, and hydrolysis, to produce enantiomer pure or enrichment.The method of measuring optical purity comprises: the chiral ester of preparing racemic mixture under the existence of alkali, for example menthyl ester, for example (-) chloro-carbonic acid menthyl ester, or Mosher ester (acetic acid α-methoxyl group-α-(trifluoromethyl) phenylester (Jacob III.J.Org.Chem., (1982) 47:4165), and analyze ¹hNMR spectrum, determines the existence of two kinds of atropisomer enantiomers or diastereomer.According to the method that separates atropisomer naphthyl-isoquinolin (WO96/15111), by just and reverse-phase chromatography, can separate and the stable diastereomer that separates atropisomer compound.By method (3); can be by separate racemic mixture (" Chiral Liquid Chromatography " (1989) W.J.Lough of two enantiomers by the chromatograph of chiral stationary phase; Ed., Chapman and Hall, New York; Okamoto, J.of Chromatogr., (1990) 513:375-378).For example, by the method (optically-active and circular dichroism) of distinguishing that other chiral molecule (with asymmetric carbon atom) uses, can distinguish enrichment or pure enantiomer.

chemotherapeutant

Some chemotherapeutant has with compound or its pharmaceutical salts of formula I and combines the character that suppresses the confirmed surprising of in vitro and in vivo cell proliferation and do not expect.Described chemotherapeutant comprises GDC-0973 and PD-0325901.

GDC-0973, also referred to as XL-518, is the selective depressant of MEK, and it is also referred to as mitogen activated protein kinase (MAPKK), and it is the key component of the RAS/RAF/MEK/ERK approach of frequent activation in human tumor.The inappropriate activation of MEK/ERK approach has promoted the Growth of Cells in the situation that not there is not the exogenous growth factor.Carrying out evaluating for solid tumor the I clinical trial phase of GDC-0973.GDC-0973 can be as prepared described in International Patent Application Publication WO2007044515 (A1).GDC-0973 has following name: (S)-(3, the fluoro-2-of 4-bis-(the fluoro-4-iodophenyl of 2-amino) phenyl) (3-hydroxyl-3-(piperidin-2-yl) azetidine-1-yl) ketone, and there is following structure:

PD-0325901 (CAS number of registration 391210-10-9, Pfizer) be the second filial generation non--the competitive allosteric mek inhibitor of ATP, it is for potential oral tablet treatment (US6960614 of cancer; US6972298; US2004/147478; US2005/085550).Carry out II clinical trial phase for breast cancer tumour, colon cancer tumor and melanomatous potential treatment.PD-0325901 called after (R)-N-(2,3-dihydroxy propoxyl group)-3, the fluoro-2-of 4-bis-(the fluoro-4-iodophenyl of 2-amino) Benzoylamide, and there is following structure:

pharmaceutical composition

Pharmaceutical composition of the present invention or preparation comprise the combination of compound or its pharmaceutical salts, chemotherapeutant and one or more pharmaceutical carriers, fluidizer, diluent or the excipient of formula I.

An example comprises the first preparation for oral delivery GDC-0068 or its salt and one or more pharmaceutical carriers, fluidizer, diluent or excipient, and for the second preparation of a kind of of oral delivery GDC-0973 and PD-0325901 or its salt and one or more pharmaceutical carriers, fluidizer, diluent or excipient.In an example, described the second preparation comprises GDC-0973 or its salt.

The compound of formula I or its pharmaceutical salts and chemotherapeutant can non-solvated form or the solvation form with medicinal solvent such as water, ethanol etc. exist, and the invention is intended to contain solvation and non-solvated form simultaneously.

Can also there is different tautomeric forms in the compound of formula I or its pharmaceutical salts and chemotherapeutant, these all forms are included in the scope of the present invention.Term " tautomer " or " tautomeric form " refer to can be by the low energy barrier constitutional isomer with different-energy of conversion mutually.For example, proton tautomerism body (also claiming protolysis tautomer (prototripic tautomer)) comprises the mutual conversion of being undertaken by the migration of proton, for example keto-enol and imines-enamine isomerization.Valence tautomerism body comprises the mutual conversion of being undertaken by the reconstruct of some bonding electronss.

Pharmaceutical composition is contained (bulk) in bulk compositions and single dose unit, it for example comprises, more than a kind of (two kinds) medical active agent, comprises chemotherapeutant and any medicinal inactive excipient, diluent, carrier or fluidizer described in compound or its pharmaceutical salts and the application of formula I.The previous described medical active agent of fixed amount can be contained in described bulk composition and each single dose unit.Described bulk composition is the material that is not yet formed as single dose unit.Exemplary dose unit is oral dosage units such as tablet, pill, capsule etc.Similarly, passing through described in the application gives method that pharmaceutical composition of the present invention treats patient and is also intended to contain and gives bulk composition and single dose unit.

Pharmaceutical composition is also contained isotope-labeled compound, and it is cited with the application identical, different, and one or more atoms are had to be different from the atomic mass conventionally found at occurring in nature or the atomic mass of mass number or the atom of mass number and to substitute.Cited any concrete atom or all isotopes of element include within the scope of the compounds of this invention and its purposes.The exemplary isotope that can be incorporated into the compounds of this invention comprises the isotope of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine and iodine, for example ²h, ³h, ¹¹c, ¹³c, ¹⁴c, ¹³n, ¹⁵n, ¹⁵o, ¹⁷o, ¹⁸o, ³²p, ³³p, ³⁵s, ¹⁸f, ³⁶cl, ¹²³i and ¹²⁵i.Some isotope-labeled the compounds of this invention (is for example used ³h and ¹⁴c labelling) can be used in compound and/or the test of substrate tissue distribution.Because it is easy to preparation and determination methods, in tritium generation, ( ³h) and carbon-14 ( ¹⁴c) isotope is useful.In addition, with higher isotope, for example deuterium is ( ²h) replacement can provide some treatment advantage, and this is for example, because it produces larger metabolic stability (, Half-life in vivo increases or dosage requires to lower), is preferred thus in some cases.Positron emission isotope for example ¹⁵o, ¹³n, ¹¹c and ¹⁸f can be effective to positron emission Imaging (PET) research, with detection substrate receptor share.

The compound of formula I or its pharmaceutical salts and chemotherapeutant comprise that according to being used for the treatment of property treatment (comprising prophylactic treatment) mammal the standard pharmaceutical of the therapeutic combination of the excess proliferative disease in the mankind puts into practice to prepare.The invention provides pharmaceutical composition, the chemotherapeutant described in the compound of its contained I or its pharmaceutical salts and one or more the application and the combination of one or more pharmaceutical carriers, fluidizer, diluent or excipient.

That suitable carrier, diluent and excipient is well known to those skilled in the art and comprise following material such as carbohydrate, wax, water solublity and/or polymers capable of swelling, hydrophilic or hydrophobic material, gelatin, oil, solvent, water etc.Concrete carrier, diluent or the excipient using depends on application process and the object of the compounds of this invention.The selection of solvent is generally thought and can safety (GRAS) be administered to mammiferous solvent based on those skilled in the art.Conventionally, safety solvent is nontoxic aqueous solvent, for example water, and other innoxious solvent that can dissolve or dissolve each other in water.Suitable aqueous solvent comprises water, ethanol, propylene glycol, Polyethylene Glycol (for example PEG400, PEG300) etc., and its mixture.Preparation can also comprise one or more buffer agents, stabilizing agent, surfactant, wetting agent, lubricant, Emulsion, suspending agent, antiseptic, antioxidant, opacifier, fluidizer, processing aid, pigment, sweeting agent, spice, flavoring agent and other known additive, the medicine (being compound of the present invention or its pharmaceutical composition) of exquisite outward appearance is provided to provide or helps preparation pharmaceutical product (being medicine).

Can prepare preparation with routine dissolving and mixed method.For example, under the existence of one or more excipient as above, bulk drug material (being the stabilized form complex of cyclodextrin derivative or other known complexometric reagent (for example, with) of compound of the present invention or compound) is dissolved in suitable solvent.The compounds of this invention is usually formulated as to pharmaceutical dosage form to be held the medicine of manageable dosage and makes patient can comply with the dosage regimen of opening to provide.

The application's pharmaceutical composition (or preparation) can be packed in every way, and this depends on the method that administration medicine uses.For example, can comprise container for the article that distribute, therein, pharmaceutical preparation is placed with suitable form.Suitable container is known for those skilled in the art, and comprises material for example bottle (plastic and glass), pouch, ampoule, plastic bag, metal cylinder etc.Container can also comprise anti-tampering system, to prevent the inclusions of use packing imprudent.In addition, container has label disposed thereon, and label is described the inclusions of container.Label can also comprise suitable warning.

The pharmaceutical preparation of compound can be prepared for various route of administration and type.For example, compound or its pharmaceutical salts with the formula I of expection purity are optionally mixed (Remington's Pharmaceutical Sciences (1995) 18th edition with medicinal diluent, carrier, excipient or stabilizing agent, Mack Publ.Co., Easton, PA), its form is powder or the aqueous solution of lyophilized formulations, grinding.Preparation can be by mixing to carry out to the avirulent carrier of receiver during in the dosage being adopted and concentration with physiology's acceptable carrier at suitable pH and with the purity of expection in ambient temperature.The pH of preparation depends primarily on the concentration of concrete purposes and compound, but its scope is approximately 3 to approximately 8.

Pharmaceutical preparation is optionally aseptic.Especially, must be aseptic for the preparation of vivo medicine-feeding.Described sterilizing can be by filtering and easily complete through aseptic filter membrane.

Pharmaceutical preparation conventionally can be used as solid composite, lyophilized formulations or aqueous solution and stores.

Dosage and administration are carried out in pharmaceutical preparation in a certain way, and quantity, concentration, progress, course, carrier and route of administration all meet good medical practice.The factor of considering in this application comprises the clinical disease of treated concrete disease, the concrete mammal for the treatment of, individual patient, the cause of disease of disease, the schedule of sending site, medication, administration of medicament, and the known other factors of medical professional." the treatment effective dose " of the compound of institute's administration depends on this consideration, and is the minimum flow that prevention, improvement or the disease for the treatment of thrombin mediation need.Described amount is preferably lower than host being produced to toxicity or making host obviously be easier to hemorrhage amount.

Acceptable diluent, carrier, excipient and stabilizing agent are avirulent for experimenter under using dosage and concentration, and comprise buffer, for example phosphate, citrate and other organic acid; Antioxidant, comprises ascorbic acid and methionine; Antiseptic (for example stearyl dimethyl benzyl ammonium chloride; Hexamethonium chloride; Benzalkonium chloride, benzethonium chloride; Phenol, butyl or benzyl alcohol; Alkyl paraben is P-hydroxybenzoic acid methyl ester or propyl parabene for example; Catechol; Resorcinol; Hexalin; 3-amylalcohol; And metacresol); Low-molecular-weight (being less than about 10 residues) polypeptide; Albumen, for example serum albumin, gel, or immunoglobulin; Hydrophilic polymer is polyvinylpyrrolidone such as; Aminoacid is glycine such as, glutamine, ASPARTIC ACID, histidine, arginine, or lysine; Monosaccharide, disaccharide and other carbohydrate, comprise glucose, mannose, or dextrin; Chelating agen is EDTA such as; Sugar is sucrose such as, mannitol, trehalose or Sorbitol; Form the counter ion of salt, for example sodium; Metal complex (for example, Zn-protein complexes); And/or such as TWEEN of nonionic surfactant ^tM, PLURONICS ^tMor Polyethylene Glycol (PEG).Active drug component can also be collected in microcapsule, microcapsule for example utilizes condensation technique or utilizes interfacial polymerization to prepare, for example hydroxy methocel or gel-microcapsule and poly-(methyl methacrylate) microcapsule, respectively for example, at the drug delivery system (liposome, albumin microsphere spheroid, micro emulsion, nano-particle and nano-microcapsules) of colloid or in coarse emulsion.This technology is to be disclosed in Remington's Pharmaceutical Sciences 18th edition, (1995) Mack Publ.Co., and Easton, in PA.

Can the compound of preparation formula I or the slow releasing preparation of its pharmaceutical salts.The suitable example of slow releasing preparation comprises the semipermeability substrate of the solid hydrophobic polymer that contains formula I compound or its pharmaceutical salts, and this substrate is formed article form, for example film or microcapsule.The example of sustained-release matrix comprises polyester, hydrogel (for example poly-(methacrylic acid 2-hydroxyethyl ester) or poly-(vinyl alcohol)), polyactide (United States Patent (USP) 3,773,919) for example LUPRON DEPOT of the copolymer of, Pidolidone and γ-ethyl-Pidolidone ester, nondegradable ethane-acetic acid ethyenyl ester, degradable poly lactic coglycolic acid ^tM((the injectable microsphere being formed by poly lactic coglycolic acid and leuprorelin acetate) and poly-D-(-)-3-hydroxybutyric acid.

Pharmaceutical preparation comprises the preparation of the route of administration that is suitable for the application's detailed description.Preparation can exist with unit dosage forms aptly and can prepare by the known any method of pharmaceutical field.Technology and preparation conventionally can be at Remington's Pharmaceutical Sciences18 ^thed. (1995) Mack Publishing Co., Easton, finds in PA.Described method comprises the step that active component and the carrier that forms one or more auxiliary agents are associated.Generally speaking, preparation is prepared by the following method: if make active component and liquid-carrier or the meticulous solid carrier separating or two kinds of carriers associate equably and closely and need to make product molding.

Be suitable for unit such as pill that the compound of formula I of oral administration or the preparation of its pharmaceutical salts and/or chemotherapeutant can be prepared as dispersion, hard or soft capsule (for example gelatine capsule), cachet, lozenge, lozenge, aqueous or oiliness suspensoid, dispersible powder or granule, Emulsion, syrup or spirit, it contains compound or its pharmaceutical salts and/or the chemotherapeutant of the formula I of the amount of pre-determining separately.As combination preparation, the amount of the compound of formula I or the amount of its pharmaceutical salts and chemotherapeutant can be formulated as pill, capsule, solution or suspensoid.Selectively, the compound of formula I or its pharmaceutical salts and chemotherapeutant can separately be formulated as pill, capsule, solution or the suspensoid for alternately administration.

Preparation can be according to preparing for the preparation of known in the art any method of pharmaceutical composition and described compositions can contain one or more reagent and comprises sweeting agent, flavoring agent, coloring agent and antiseptic, so that agreeable to the taste preparation to be provided.Compressed tablets can be prepared such as powder or granule (its optional and binding agent, lubricant, inert diluent, antiseptic, surfactant or dispersant) by active component being compressed to free-pouring form in suitable machine.Molded tablet can by suitable machine by molded preparation of mixture of the active component by the moistening powdered of inert liquid diluent.

Tablet can be optionally provided active component from wherein slowly or control and discharge by coating or indentation and optional preparation thus.The tablet excipient of pharmaceutical preparation can comprise: filler (or diluent), and it increases the cumulative volume of the powdered medicine that forms tablet; Disintegrating agent, it promotes that disintegration of tablet is fractionlet in the time taking in, and is desirably single medicine granule, and promotes quick dissolving and the absorption of medicine; Binding agent, it guarantees that granule and tablet can form afterwards and have required mechanical strength and keep together with tablet in compacting, this prevents that it is split into its component powders in packing, shipment and routine operation process; Fluidizer, the powder of its improvement formation tablet mobility in process of production; Lubricant, it guarantees that tabletting powder does not adhere to the instrument for compressed tablets in manufacture process, it has improved mixture of powders through the mobile of press and in the time that the tablet completing is sprayed by instrument, has made friction and break to minimize; Antitack agent, it has the function that is similar to fluidizer, and it is reduced in and in manufacture process, forms the powder of tablet and for the adhesion between the machine of compressed tablets; Correctives, it mixes in tablet suitable taste to be provided or to shelter unfavorable taste; And coloring agent, it contributes to distinguish the compliance with patient.

Containing active component is acceptable with the tablet of the mixture that is suitable for the nontoxic pharmaceutical excipient of manufacturing tablet.These excipient can be for example inert diluent, such as calcium carbonate or sodium carbonate, lactose, calcium phosphate or sodium carbonate; Granulating agent and disintegrating agent are such as corn starch or alginic acid; Binding agent such as starch, gelatin or arabic gum; And lubricant such as magnesium stearate, stearic acid or Pulvis Talci.Tablet formulation can be without coating or comprise microencapsulation coating by known technology, to delay in gastrointestinal tract disintegrate and absorption and the continuous action that lasts the long term be provided thus.For example, can adopt the combination of time dilation material such as independent glyceryl monostearate or distearin or itself and wax.

For treatment eye or other outside organization for example mouth and skin, preparation is preferably used as topical ointments or ointment, the active component of its amount that contains for example 0.075-20%w/w.In the time being formulated as ointment, active component can adopt together with paraffin or the mixable ointment base of water.Selectively, active component can be formulated as the ointment with oil in water emulsion substrate.

If needed, the water of emulsifiable paste matrix can comprise polyhydric alcohol, has alcohol such as propylene glycol, fourth-1 of two or more hydroxyl, 3-glycol, mannitol, sorbitol, glycerol and Polyethylene Glycol (comprising PEG400) and their mixture.Topical formulations can expectedly comprise enhanced activity composition through skin or other relevant range absorbs or the compound of infiltration.The example of described transdermal penetration reinforcing agent comprises dimethyl sulfoxide and related analogs.

The oil phase of Emulsion of the present invention can be made up of in known manner principal component, comprises at least one emulsifying agent and fat or oily mixture, or at least one emulsifying agent and fat and oily mixture.Preferably, hydrophilic emulsifier is included with together with lipophilic emulsifier as stabilizing agent.Emulsifying agent with/do not form emulsifing wax together with stabilizing agent, and described wax forms emulsifying ointment base together with oil & fat, it forms the oily decentralized photo of cream preparation.The emulsifying agent and the Emulsion stabilizing agent that are applicable to described preparation comprise

cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl monostearate and sodium laurylsulfate.

The aqueous suspension of pharmaceutical preparation contains active substance and the mixture that is suitable for the excipient of manufacturing aqueous suspension.Described excipient comprises suspending agent, such as sodium carboxymethyl cellulose, croscarmellose, polyvidone, methylcellulose, hydroxypropyl emthylcellulose, sodium alginate, polyethylene-ketopyrrolidine, Tragacanth and arabic gum; Dispersant or wetting agent, for example, for example, for example, such as the condensation product (heptadecane oxirane spermol) of the condensation product of naturally occurring phospholipid (lecithin), oxyalkylene and fatty acid (poly-stearic acid oxygen vinyl acetate), ethylene oxide and long chain aliphatic, ethylene oxide and for example, derived from the condensation product (polyoxyethylene sorbitan monooleate) of the partial ester of fatty acid and hexitan.Aqueous suspension can also contain one or more antiseptic (for example P-hydroxybenzoic acid ethyl ester or propyl p-hydroxybenzoate), one or more coloring agent, one or more flavoring agents and one or more sweeting agents (for example sucrose or glucide).

Pharmaceutical composition can be aseptic injection preparation form, for example sterile injectable moisture or containing oil suspension.Can prepare this suspension according to methods known in the art, use above-mentioned those suitable dispersions or wetting agent and suspending agent.Aseptic injection preparation can also be can accept solution or the suspension in diluent or solvent at nontoxic parenteral, for example solution in 1,3 butylene glycol, or be prepared into lyophilization powder form.Among acceptable carrier and solvent, operable is water, Ringer's solution and isotonic sodium chlorrde solution.In addition, can use traditionally aseptic expressed oi as solvent or suspension media.For this object, can use any soft expressed oi, comprise synthetic list or two glyceride.In addition, also can in ejection preparation, use such as oleic acid of fatty acid.

The amount that can mix with carrier mass the active component that produces single dosage form will depend on that subject host changes with concrete administering mode.For example, be intended to be administered orally in the mankind time m-delivery formulations can be containing the active substance of the 1-1000mg that has an appointment, itself and carrier mass suitable and amount easily are compound, and described amount can change between Overall Group's compound (weight: weight) of approximately 5 to approximately 95%.Can pharmaceutical compositions so that the amount that is easy to the administration of measuring to be provided.For example, the active component that the aqueous solution that is intended to intravenous infusion can contain the about 3-500 μ of every ml soln g is to make to produce the infusion with the proper volume of the speed of about 30mL/hr.

The preparation that is suitable for parenteral comprises moisture and anhydrous aseptic parenteral solution, and it can contain antioxidant, buffer, antibacterial and make preparation and solute that predetermined experimenter's blood etc. oozes; With the aseptic suspension of moisture and non-water, it can comprise suspending agent and thickening agent.

Be suitable for also comprising eye drop to the preparation of eye topical, wherein active component be dissolved in or be suspended in suitable carrier, especially for the aqueous solvent of active component.Described active component is preferably present in described preparation with for example concentration of about 0.5-10%w/w, about 1.5%w/w of about 0.5-20%w/w.

Be suitable for the preparation of topical in mouth and comprise lozenge, it is included in the active component in taste masking substrate, and described substrate is generally sucrose and arabic gum or tragakanta; Pastille, it is included in the active component in inert base, and described substrate is such as gelatin and glycerol, or sucrose and arabic gum; And collutory, it is included in the active component in suitable liquid-carrier.

Can be used as the suppository existence with suitable substrate for the preparation of rectally, described substrate comprises for example cocoa butter or Salicylate.

Be suitable in lung or granularity that the preparation of nose administration has in 0.1 to 500 micrometer range for example (comprises the granularity in the middle of 0.1 and 500 micrometer ranges, increment micron is for example 0.5,1,30 micron, 35 microns etc.), carry out administration by making it suck fast nasal cavity, or carry out administration by sucking oral cavity, to reach in alveolar sac.Suitable preparation comprises aqueous or the oily solution of active component.The preparation that is suitable for aerosol or dry powder administration can be prepared according to conventional method, and can for example be used for the treatment of so far with other therapeutic agent or prevent to send together with the compound of disease as described below.

The preparation that is suitable for vagina administration can provide with the form of vaginal suppository, plug, emulsifiable paste, gel, paste, foam or spray, except containing active component, also contains suitable carrier known in the art.

Preparation can also be packaged in single dose or multi-dose container, for example ampoule and the bottle of sealing, and can be kept under lyophilization (lyophilizing) condition, only need to before being about to use, add the aseptic liquid-carrier of injection, for example water.Interim injection and suspension are to prepare with the sterilized powder of previously described kind, granule and tablet.Preferred unit dose formulations is to contain daily dose or the unit day sub-doses of above-named active component herein, or those preparations of its suitable part.

The present invention also provides veterinary composition, the compound of its contained I or its pharmaceutical salts and at least one chemotherapeutant and carrier for animals as defined above.Carrier for animals is the material that can be used for administration composition object, and can be solid, liquid or gaseous material, its be inertia or be acceptable in veterinary applications, and compatible with active component.These veterinary compositions can parenteral, oral administration, or by any other required administration.

combined therapy

The compound of formula I or its pharmaceutical salts can be used for the treatment of excess proliferative disease or disease comprise tumor, cancer and neoplasia tissue and worsen before and other chemotherapeutant of non-neoplasm or non-malignant excess proliferative disease be used in combination.In certain embodiments, in the dosage regimen as combined therapy, make the compound of formula I or its pharmaceutical salts and there is anti-hyper-proliferative character or be used for the treatment of the second compound combination of excess proliferative disease.The second compound of dosage regimen preferably has and the compound of formula I or the activity of its pharmaceutical salts complementation, and they can adversely not affected each other.Described compound can effectively be measured and give for expection object.In one embodiment, give therapeutic combination by dosage regimen, in described dosage regimen, the compound of formula I for the treatment of effective dose or its pharmaceutical salts once gave in the scope of (q3wk) twice to every three weeks of every day, and the chemotherapeutant for the treatment of effective dose gave for twice in every day to every triweekly scope.

Combined therapy can be used as simultaneously or priority dosage regimen gives.In the time successively giving, described combination can twice or multiple dosing give.Combination medicine-feeding comprises the co-administered that uses separate formulation, and with the successive administration of any order, wherein preferably exists two kinds of (or all) active component to show their bioactive a period of time simultaneously.

Of the present invention one concrete aspect, the compound of formula I or its pharmaceutical salts can be after starting to give one or more medicines the administration time period of approximately 1 to approximately 10 day.Of the present invention another concrete aspect, the compound of formula I or its pharmaceutical salts can be before starting to give described combination the administration time period of approximately 1 to approximately 10 day.Of the present invention another concrete aspect, the administration of the compound of formula I or the administration of its pharmaceutical salts and chemotherapeutant is starting on the same day.

For the suitable dosage of any medicine jointly giving be above use at present those and can reduce, this is because of the new medicine of differentiating and the compound action (synergism) of other chemotherapeutant or treatment, such as increasing therapeutic index or alleviating toxicity or other side effect or consequence.

In the specific embodiments of anticancer therapy, the compound of formula I or its pharmaceutical salts can combine with chemotherapeutant, and combine with surgical intervention and X-ray therapy.By the compound of selecting type I or its pharmaceutical salts and the amount of other medical active chemotherapeutant and the relevant time limit of administration to realize the combined therapy effect of expection.

the administration of pharmaceutical composition

Compound can be suitable for sanatory administration by any.That suitable approach comprises is oral, in parenteral (comprise in subcutaneous, intramuscular injection, intravenous, intra-arterial injection, suction, intradermal, sheath, epidural and infusion techniques), transdermal, rectum, per nasal, part (comprising buccal and Sublingual), vagina, intraperitoneal, lung and intranasal administration.Topical also can relate to the use of transdermal administration such as transdermal patch or Iontophoretic device.

Pharmaceutical preparation is in Remington's Pharmaceutical Sciences, 18 ^thed., (1995) Mack Publishing Co., Easton, discusses in PA.Other example of pharmaceutical preparation can be at Liberman, H.A.and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, Vol3,2 ^nded., New York, finds in NY.For local immunosuppression treatment, compound can pass through intralesional administration, comprises perfusion or otherwise before transplanting, makes graft contact with inhibitor.Should be realized that preferred approach can change along with for example receiver's situation.When by compound oral administration, it can be formulated as pill, capsule, tablet etc. together with pharmaceutical carrier, fluidizer or excipient.When by compound parenteral, it can be prepared with medicinal the intestines and stomach Jie of western medium thing or diluent, and it is unit dose injectable forms as detailed below.

The dosage range for the treatment of human patients can be extremely compound or its pharmaceutical salts of the formula I of about 1600mg of about 20mg every day.Typical dosage can be the compound of about 50mg to about 800mg.Dosage can give once (QD), every day give (BID) for twice or give more continually every day, pharmacokinetics (PK) and pharmacodynamics (PD) character that this depends on particular compound, comprise absorption, distribution, metabolism and excretion.In addition, toxicity considerations will can affect dosage and dosage regimen.In the time of oral administration, pill, capsule or tablet can twice absorptions every day, take in once a day or more do not take in continually such as once in a week or once every two weeks or within every three weeks, once take in the time period that reaches appointment.Can repeat described dosage regimen for numerous cycles for the treatment of.

therapeutic Method

(1) therapeutic combination of the compound of formula I or its pharmaceutical salts and (2) chemotherapeutant is used for the treatment of disease, disease and/or obstacle, include but not limited in mammal regulated by AKT kinases those.Can include but not limited to mesothelioma, carcinoma of endometrium, glioma, cancer of pancreas, breast carcinoma, pulmonary carcinoma, ovarian cancer, carcinoma of prostate, melanoma, gastric cancer, colon cancer, head and neck cancer according to the cancer of the inventive method treatment.

Determine that some combination of the present invention provides the effect for the improvement of some cancerous phenotype.For example, some combination of the present invention provides the effect for the improvement of the cancer relevant to PTEN sudden change (or state of low or nothing), AKT sudden change (or high pAKT expresses or level of amplification), PI3K sudden change, Her2/ErbB2 amplification, RAS sudden change, RAF sudden change or combinations thereof.

Therefore, the combination of some described in the application can be effective especially for the cancer of these types.

For example, in colorectal carcinoma, estimate that the combination of PI3k/AKT sudden change (for example PI3K H1047R, E545K, D549N, P421L, L568F, L569F, P449T or their combination) and RAS/RAF sudden change (KRAS G13D, G12D, G12V or their combination) is combined and replied by force and observe strong synergism for the combination of GDC-0068 and GDC-0973 the application.

Equally, in nonsmall-cell lung cancer, can be observed strong synergism for the combination of GDC-0068 and GDC-0973, wherein: (i) have the combination of PI3k/AKT sudden change (PI3k E545K, L997P, M772X, N996H or their combination) and RAS/RAF sudden change (Q61H, G12C, Q61K, N85K, G12S, BRAF V600E or their combination), and (ii) have the combination of the RAS/RAF without PI3k sudden change.

Equally, in melanoma, can be observed strong synergism for the combination of GDC-0068 and GDC-0973, wherein: (i) have BRAF V600E sudden change, and (ii) have the BRAF V600E sudden change or disappearance or the amplification that there is PTEN sudden change (nothing or low state) or there is high pAKT expression or activity level.

The test kit whether for test patient with BRAF V600E sudden change is what be available commercially.An example is 4800BRAF V600 sudden change test (RocheMolecularSystems Inc.), detects BRAF V600E sudden change in its paraffin-embedded (FFPET) mankind melanoma tissue of fixing at formaldehyde.It has been ratified as the combination diagnosis (companion diagnostic) for Wei Luofeini (vemurafenib) or the treatment of its pharmaceutical salts in the U.S., and it is designed to treat the wherein patient of the mutant form of the hiding BRAF gene of melanoma.Before clinical and in clinical research,

bRAF sudden change test has 97.3% positive concordance in detection BRAF V600E (1799T>A) sudden change, and these all BRAF sudden changes that represent >～85% are reported in COSMIC data base.

In the paraffin-embedded tissue (FFPET) of fixing at formaldehyde,

bRAF sudden change test can detect the V600E sudden change of >5% sudden change level.This test also can detect other V600 and suddenly change such as V600D and V600K.

bRAF sudden change test can be carrying out in <8 hour from receiving sample (such as the tissue sample being obtained by patient or tumor cell).

4800BRAF V600 sudden change test for 4800 systems, the PCR in real time test on v2.0, and it is intended to for the assisted Selection melanoma patients that wherein tumor is suddenlyd change with BRAFV600E.

PTEN can measure by any proper method known in the art without (or low) state.In an example, use IHC.Selectively, can use western blot analysis.Be (Cell Signaling Technology, Beverly, MA, Cascade Biosciences, Winchester, the MA) being available commercially to the antibody of PTEN.Be described in Neshat for the IHC of PTEN state and the exemplary operation of western blot analysis, M.S.et al.Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR, Proc.Natl Acad.Sci.USA98, 10314 – 10319 (2001) and Perren, A., et.al.Immunohistochemical Evidence of Loss of PTEN Expression in Primary Ductal Adenocarcinomas of the Breast, American Journal of Pathology, Vol.155, No.4, in October1999.In addition can differentiate by technology known in the art to AKT sudden change, PI3K sudden change and the Her2/ErbB2 relevant cancer that increases.

The level of the activation of AKT or phosphorylation in given sample (" pAKT ") can be measured by methods known in the art than disactivation or unphosphorylated AKT level.PAKT state proportionally (for example the amount of the pAKT in tumor cell is divided by the amount of the pAKT in the non-tumor cell of same type) or subtraction (for example amount of the pAKT in tumor cell deducts the amount of the pAKT in the non-tumor cell of same type) represents.PAKT distributes and also can represent as follows: the level of activated channel for example, obtaining by measuring the amount of phosphorylation downstream targets (pGSK or PRAS40) of AKT.High pAKT distributes and refers to that the activation of whole AKT in sample or phosphorylation level are higher than baseline value.In an example, described baseline value is the basic horizontal for the pAKT of given cell type.In other example, described baseline value is at given sample cell colony for example meansigma methods or the average level of the pAKT in non-cancerous cells.In other example, high pAKT refers to for example, meansigma methods when normal, healthy (non-tumor) cell than the same type from identical mammal or patient colony, crosses the tumor cell of the AKT of expression or amplification phosphorylation or activation in cell.Described pAKT distributes and for example also can be used for, with other labelling (FOXO3a location distributes) combination to predict the usefulness of some PI3k/AKT kinase pathways inhibitor, or the usefulness of being for example combined with BRAF V600E mutation status with some combination of prediction type I compound and Wei Luofeini, particularly the cancer with Wei Luofeini opposing such as transitivity or unresectable melanomatous patient in.Be (for example phosphorus-Akt (Thr308) STAR ELISA test kit, the EMD Millipore) being available commercially for the test kit of measuring tissue sample pAKT.

Be (Qiagen) being available commercially for the test kit of testing PI3k, KRAS and AKT sudden change existence.

One concrete aspect, the invention provides to be used for the treatment of and have to PTEN sudden change or express loss, AKT sudden change or amplification, PI3K sudden change or amplification, Her2/ErbB2 sudden change or amplification, KRAS sudden change or amplification, BRAF sudden change or amplification or the patient's of the cancer that their combination is relevant method, comprise to described patient and combining.In yet another aspect, the invention provides for differentiating that patient has the method for the cancer of available combined therapy of the present invention, comprise whether the cancer of determining described patient loses with PTEN sudden change or expression, AKT sudden change or amplification, PI3K sudden change or amplification or Her2/ErbB2 amplification, KRAS sudden change or amplification, BRAF sudden change or amplification or their combination are relevant, wherein said patient's cancer and PTEN sudden change or expression loss, AKT sudden change or amplification, PI3K sudden change or amplification or Her2/ErbB2 amplification, KRAS sudden change or amplification, the measurable described cancer of dependency of BRAF sudden change or amplification or their combination can be used combined therapy of the present invention.In yet another aspect, the invention provides method, it further comprises the patient who so differentiates with combined therapy of the present invention.In one embodiment, described cancer is ovarian cancer, breast carcinoma, melanoma, colon cancer or nonsmall-cell lung cancer.

goods

In other embodiments of the present invention, the compound that contains the formula I that is used for the treatment of disease as above and disease or goods or " test kit " of its pharmaceutical salts are provided.In one embodiment, described test kit comprises compound or its pharmaceutical salts of container and formula I.

Test kit can further comprise relatively label or package insert on container or with container.Term " package insert " refers to the explanation in the commercial packing that is usually included in treatment product, the introduction that it comprises related index, purposes, dosage, administration, the contraindication of this treatment product and/or relates to the warning of use.Suitable container comprises for example bottle, bottle, syringe, blister package etc.Container can be formed by for example glass of various materials or plastics.Container can hold compound or its pharmaceutical salts or its preparation of the formula I of effective sanatory amount, and can there is aseptic entrance (for example, container can be or to have the bottle of stopper with the intravenous solution bag of subcutaneous injection needle-penetration).At least one activating agent in compositions is compound or its pharmaceutical salts of formula I.Label or package insert indicate described compositions and are used for the treatment of selected disease such as cancer.In one embodiment, label or package insert indicate the compound of contained I or the compositions of its pharmaceutical salts and can be used for the disease that treatment is caused by abnormal cell growth.Label or package insert also can indicate described compositions and can be used for treating other disease.Selectively or extraly, goods can further comprise second container, it comprises for example bacteriostatic water for injection of medicinal buffer (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.It can further comprise other desirable material (from commodity and user viewpoint), comprises other buffer, diluent, filter, pin and syringe.

Test kit can further comprise the compound of Medicine-feeding type I or the indication of its pharmaceutical salts and the second pharmaceutical preparation (if present).For example, if test kit comprises the first compositions (compound of contained I or its pharmaceutical salts and the second pharmaceutical preparation), test kit can further comprise while, priority or the separately indication of patient's first and second pharmaceutical compositions of administration needs.

In other embodiments, test kit is suitable for compound or its pharmaceutical salts of the formula I of the oral form of delivery of solids, for example tablet or capsule.Preferably, this test kit comprises many unit dose.This test kit can comprise card, and it has the dosage that uses order orientation with their expection.The example of this test kit is " blister package ".Blister package is well known in packaging industry, and is widely used for packaged pharmaceuticals unit dosage forms.If necessary, can provide memory aid, for example, with number, letter or other mark form, or with schedule, at treatment schedule acceptance of the bid number tomorrow that can dosage.

According to an embodiment, test kit can comprise (a) first container, has the compound or its pharmaceutical salts that contain formula I therein; With optional (b) second container, there is the second pharmaceutical preparation containing therein, wherein the second pharmaceutical preparation comprises second compound with anti-hyper-proliferative activity.Alternatively or additionally, test kit can further comprise the 3rd container, it comprises for example bacteriostatic water for injection of medicinal buffer (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.It can further comprise other desirable material (from commodity and user viewpoint), comprises other buffer, diluent, filter, pin and syringe.

In the time that the compound of the contained I of test kit or its pharmaceutical salts and the second therapeutic agent are the compositions of chemotherapeutant, test kit can comprise container, it is for holding independent compositions, the bottle for example separating or the thin foil packing of separating, but, within independent compositions can also be included in single undivided container.Conventionally the instructions that, test kit comprises the independent component of administration.When preferably for example, with the component of separating of different dosage form (oral and parenteral) administration, be with different spacing of doses administrations, or in the time that prescriber wants the dosage of the one-component of determining coupling medicine, kit form is particularly advantageous.

concrete aspect of the present invention

Of the present invention one concrete aspect, excess proliferative disease is cancer.

Of the present invention one concrete aspect, cancer and PTEN sudden change is relevant.

Of the present invention one concrete aspect, cancer and AKT sudden change, cross express or amplification relevant.

Of the present invention one concrete aspect, cancer and PI3K sudden change is relevant.

Of the present invention one concrete aspect, cancer and KRAS sudden change is relevant.

Of the present invention one concrete aspect, cancer and BRAF sudden change is relevant.

Of the present invention one concrete aspect, cancer and (1) PTEN, AKT or PI3K sudden change are relevant with the combination of (2) KRAS or BRAF sudden change.In an example, cancer is ovarian cancer, breast carcinoma, melanoma, colon cancer or nonsmall-cell lung cancer.

Of the present invention one concrete aspect, cancer is to a kind of or two kinds of opposings in the treatment of GDC-0068 and GDC-0973 single medicine, but the combined therapy of GDC-0068 and GDC-0973 is replied.In an example, cancer is ovarian cancer, breast carcinoma, melanoma, colon cancer or nonsmall-cell lung cancer.

Of the present invention one concrete aspect, cancer is selected from mesothelioma, carcinoma of endometrium, cancer of pancreas, breast carcinoma, pulmonary carcinoma, ovarian cancer, carcinoma of prostate (for example carcinoma of prostate of castrating opposing), melanoma, gastric cancer, colon cancer, renal carcinoma, head and neck cancer and glioma.

Of the present invention one concrete aspect, the compound of oral giving construction I or its pharmaceutical salts.

Of the present invention one concrete aspect, the combination of the compound of giving construction I or its pharmaceutical salts and GDC-0973 or PD-0325901 or its pharmaceutical salts.

Of the present invention one concrete aspect, the combination of the compound of giving construction I or its pharmaceutical salts and GDC-0973 or PD-0325901 or its pharmaceutical salts and cancer are cancer of pancreas.

Of the present invention one concrete aspect, the combination of the compound of giving construction I or its pharmaceutical salts and GDC-0973 or its pharmaceutical salts and cancer are cancer of pancreas.

Of the present invention one concrete aspect, the combination of the compound of giving construction I or its pharmaceutical salts and GDC-0973 or PD-0325901 or its pharmaceutical salts and cancer are colon cancer.

Of the present invention one concrete aspect, the combination of the compound of giving construction I or its pharmaceutical salts and GDC-0973 or PD-0325901 or its pharmaceutical salts and cancer are breast carcinoma.

Of the present invention one concrete aspect, the combination of the compound of giving construction I or its pharmaceutical salts and GDC-0973 or PD-0325901 or its pharmaceutical salts and cancer are ovarian cancer.

Of the present invention one concrete aspect, the combination of the compound of giving construction I or its pharmaceutical salts and GDC-0973 or PD-0325901 or its pharmaceutical salts and cancer are pulmonary carcinoma.

Of the present invention one concrete aspect, the combination of the compound of giving construction I or its pharmaceutical salts and GDC-0973 or PD-0325901 or its pharmaceutical salts and cancer are melanoma.

Of the present invention one concrete aspect, the compound of formula I or its pharmaceutical salts are formulated as to tablet.

embodiment

For example explanation the present invention, the present invention includes following embodiment.It should be understood, however, that these embodiment do not limit the present invention and are only intended to propose to put into practice method of the present invention.

Embodiment 1

(S)-2-(4-chlorphenyl)-1-(4-((5R, 7R)-7-hydroxy-5-methyl base-6,7-dihydro-5H-cyclopenta [d] pyrimidine-4-yl) piperazine-1-yl)-3-(isopropylamino) third-1-ketone

step 1: the solution in EtOAc (900mL) is cooled to-78 ℃ by pulegenic acid ethyl ester (ethyl pulegenate) (130g, 662mmol) to use dry ice-isopropanol bath.Make this mixture carry out ozone decomposed until reactant mixture becomes purple.Now, ozone stops, and reactant mixture is removed by the dry ice bath.Make bubble oxygen process reactant mixture until it becomes yellow.By reactant mixture vacuum concentration, and the residue of gained is dissolved in glacial acetic acid (400mL).Solution is cooled to 0 ℃, and lasts 30 minutes and add zinc powder (65g, 993mmol) in batches.Then reactant mixture is stirred 2 hours, now reactant mixture filtration over celite filler is removed to zinc powder.By aqueous NaOH and NaHCO for acetic acid ₃neutralization is for pH7 and use ether (3X800mL) extraction.By saline, the MgSO for Organic substance that merge ₄be dried and concentrate and obtain (2R)-2-methyl-5-oxo-cyclopentane-carboxylic acid, ethyl ester, it is brown liquid (107g, 95%).

step 2: ammonium acetate (240.03g, 3113.9mmol) is added in the solution of (R)-2-methyl-5-oxo-cyclopentane carboxylic acid, ethyl ester (106.0g, 622.78mmol) in MeOH (1.2L).Reactant mixture is stirred 20 hours in room temperature under nitrogen, judge that through TLC and HPLC it completes afterwards.Reactant mixture is concentrated, remove MeOH.The residue of gained is dissolved in DCM, uses H ₂o washed twice, uses salt water washing once, dry (Na ₂sO ₄), filtering, and concentrated obtain (R)-2-amino-5-methyl ring penta-1-olefinic carboxylic acid ethyl ester (102g, 97% yield), it is orange.LC/MS(APCI+)m/z170[M+H]+.

step 3: will contain (R)-2-amino-5-methyl ring penta-1-olefinic carboxylic acid ethyl ester (161.61g, 955.024mmol) and ammonium formate (90.3298g, 1432.54mmol) solution in Methanamide (303.456ml, 7640.19mmol) is heated to the internal temperature of 150 ℃ and stirs 17 hours.Reactant mixture is cooling, and be transferred in the mono-neck flask of 2L.Then remove excessive carbonamidine through high vacuum distillation.Once carbonamidine stops producing, the residue grease in boiler is dissolved in DCM and uses salt water washing (3X200mL).The aqueous cleaning solution of merging is extracted with DCM.By dry the organic extract merging (Na ₂sO ₄), filter and concentrate.The brown oil of gained is dissolved in minimum DCM, and uses separatory funnel this solution to be added in the solution (ether/DCM solution of about 5vol) of the stirring of ether, this causes some brown precipitate things of formation.This brown precipitate thing is filtered and removed through medium frit funnel, used ether drip washing and process.Filtrate is concentrated, grind and repeat more than twice by ether, then dry (R)-5-methyl-6,7-dihydro-5H-cyclopenta [d] pyrimidine-4-alcohol (93.225g of obtaining on fine vacuum line, 65.00% yield), it is brown color pasty solid.LC/MS(APCI-)m/z149.2.

step 4: by pure POCl ₃(463.9ml, 5067mmol) is slowly added to (R)-5-methyl-6 by addition funnel, the 0 ℃ solution of 7-dihydro-5H-cyclopenta [d] pyrimidine-4-alcohol (152.2g, 1013mmol) in DCE (1.2L).After having added, reactant mixture is warmed to room temperature, then reflux and stirring 70 minutes.Determine and reacted through HPLC.Reactant mixture is cooled to room temperature, and by excessive POCl ₃with 4 parts of following cancellation: reactant mixture is transferred in separatory funnel and drops to cooling ice and the saturated NaHCO of containing in ice bath ₃in the beaker of solution.Once the reactant mixture of each several part has added, the mixture of cancellation is stirred 30 minutes, guarantee POCl before being transferred to separatory funnel ₃destruction completely.Mixture is transferred in separatory funnel and uses DCM extracting twice.By dry the extract merging (Na ₂sO ₄), filter and concentrate.By thick material following purification on silica gel: by silica gel (1kg) hexane at 9:1 in 3L frit funnel: make slurry in ethyl acetate, silica gel vacuum is left standstill, push up with sand seal.Thick material is written into by DCM/ hexanes mixtures, and use 1L side-neck flask under vacuum by compound eluting.First high Rf by-product elutes, and then elutes the chloro-5-of (R)-4-methyl-6,7-dihydro-5H-cyclopenta [d] pyrimidine (104.4g, 61.09% yield), and it is brown oil.By triethylamine (93.0ml, 534mmol) and piperazine-1-carboxylic acid tert-butyl ester (34.8g, 187mmol) be added to the chloro-5-of (R)-4-methyl-6, in the solution of 7-dihydro-5H-cyclopenta [d] pyrimidine (30.0g, 178mmol) in n-BuOH (250mL).Reactant mixture reflux under nitrogen is also stirred and spent the night (17 hours), afterwards that it is concentrated on Rotavap.The grease of gained is dissolved in DCM, uses H ₂o washing, dry (Na ₂sO ₄), filter and concentrate.By the brown oil of gained on silica gel purification (first use the hexane of 2:1: eluent ethyl acetate until product elute neatly, then use the DCM of gradient 1:1-1:5: eluent ethyl acetate) obtain (R)-4-(5-methyl-6,7-dihydro-5H-cyclopenta [d] pyrimidine-4-yl) piperazine-1-carboxylic acid tert-butyl ester (42.0g, 74.1% yield), it is cream-coloured powder.LC/MS(APCI+)m/z319.1[M+H]+.

step 5: by solid 77%max.MCPBA (23.9g, 107mmol) be added to (R)-4-(5-methyl-6 in batches, 7-dihydro-5H-cyclopenta [d] pyrimidine-4-yl) piperazine-1-carboxylic acid tert-butyl ester (20.0g, 62.8mmol) is at CHCl ₃(310mL) in 0 ℃ of solution in.Reactant mixture is stirred 5 minutes, be then warmed to room temperature and stir 90 minutes.After 7.5 hours, observe similar HPLC.Reactant mixture is cooled to 0 ℃, then adds NaHCO ₃the m-CPBA of (13.2g, 157mmol) and other 0.5 equivalent.Reactant mixture is stirred to spend the night (14 hours).Reactant mixture is cooled to 0 ℃, and dropwise adds Na through addition funnel ₂s ₂o ₃(29.8g, 188mmol) is at H ₂solution in O (50mL).Add Na through addition funnel afterwards ₂cO ₃(24.6g, 232mmol) is at H ₂solution (mixture becomes uniformly) in O (70mL).Reactant mixture is stirred 30 minutes, then by mixture CHCl ₃(3X150mL) extraction.By dry the extract merging (Na ₂sO ₄), filter, and the concentrated N-oxide that obtains.LC/MS(APCI+)m/z335.1[M+H]+.

step 6: by Ac ₂o (77.0ml, 816mmol) is added in the N-oxide (21.0g, 62.8mmol) of step 5.Reactant mixture is heated under nitrogen in 90 ℃ of sand-baths and stir 100 minutes.Reactant mixture is cooled to room temperature, and removes excessive acetic anhydride through rotary evaporation.The grease of gained is dissolved in DCM, then pours carefully it into Na that ice is saturated ₂cO ₃in.Mixture is extracted with DCM, and by dry the extract merging (Na ₂sO ₄), filtering, and concentrated obtain (5R)-4-(7-acetoxyl group-5-methyl-6,7-dihydro-5H-cyclopenta [d] pyrimidine-4-yl) piperazine-1-carboxylic acid tert-butyl ester (23.6g, 100%), it is brown foam.LC/MS(APCI+)m/z377.1[M+H]+.

step 7: by LiOH-H ₂o (6.577g, 156.7mmol) be added to (5R)-4-(7-acetoxyl group-5-methyl-6,7-dihydro-5H-cyclopenta [d] pyrimidine-4-yl) piperazine-1-carboxylic acid tert-butyl ester (23.6g, 62.69mmol) is at the THF:H of 2:1 ₂in 0 ℃ of solution in O (320mL).Reactant mixture is stirred 10 minutes, be then warmed to room temperature.Observed identical LC/MS at 3 hours and 4.5 hours.Reactant mixture is cooled to 0 ℃, then by saturated NH ₄cl is added in mixture.Mixture is stirred 5 minutes, its most of THF is removed through rotary evaporation.By EtOAc for mixture (3X250mL) extraction, and by dry the extract merging (Na ₂sO ₄), filter, and concentrated.Thick material is carried out on Biotage65M to purified by flash chromatography (DCM of 4:1: ethyl acetate, the DCM that then gradient is 1:1-1:4: ethyl acetate).Once product eluting, crosses post by ethyl acetate rinse.Then use the remaining product of DCM:MeOH eluting (8.83g) of 30:1.Use the same terms to re-start purified by flash chromatography with Biotage40M the fraction of mixture and obtain other 2.99g, this obtains combining (5R)-4-(7-hydroxy-5-methyl base-6 of yield, 7-dihydro-5H-cyclopenta [d] pyrimidine-4-yl) piperazine-1-carboxylic acid tert-butyl ester (11.82g, 56.38% yield), it is brown foam.LC/MS(APCI+)m/z335.1[M+H]+.

step 8: the solution by DMSO (5.45ml, 76.8mmol) in DCM (50mL) is dropwise added in-78 ℃ of solution of oxalyl chloride (3.35ml, 38.4mmol) in DCM (150mL) through addition funnel.Reactant mixture is stirred 35 minutes, then slowly add (5R)-4-(7-hydroxy-5-methyl base-6 through addition funnel, 7-dihydro-5H-cyclopenta [d] pyrimidine-4-yl) solution of piperazine-1-carboxylic acid tert-butyl ester (9.17g, 27.4mmol) in DCM (80mL).Reactant mixture is stirred other 1 hour at-78 ℃, afterwards pure triethylamine (18.0ml, 129mmol) is added in mixture.Then reactant mixture is warmed to room temperature, is then stirred 30 minutes.Add H ₂o.By DCM for mixture (3X200mL) extraction, and by dry the extract merging (Na ₂sO ₄), filter, and vacuum concentration.Thick material (is rinsed post at the upper purification of silica gel (Biotage65M) with the DCM:EtOAc of about 800mL4:1, then the DCM that gradient is 1:1: eluent ethyl acetate until product elute, then use the DCM:EtOAc eluted product of 1:4) obtain (R)-4-(5-methyl-7-oxo-6,7-dihydro-5H-cyclopenta [d] pyrimidine-4-yl) piperazine-1-carboxylic acid tert-butyl ester (7.5g, 82.3% yield), it is brown foam.Foam, by DCM/ hexane concentrated (3X), is obtained to extremely shallow brown foam.HPLC>95% area.LC/MS(APCI+)m/z333[M+H]+.

step 9: by triethylamine (4.33ml, 31.1mmol; Use before use nitrogen degassed 30 minutes) and formic acid (1.36ml, 36.1mmol; Use before use nitrogen degassed 30 minutes) be added to (R)-4-(5-methyl-7-oxo-6,7-dihydro-5H-cyclopenta [d] pyrimidine-4-yl) piperazine-1-carboxylic acid tert-butyl ester (9.75g, 29.3mmol) is at DCM (210mL; Use before use nitrogen degassed 30 minutes) in solution in.Mixture is stirred 5 minutes, then add Ru catalyst (0.0933g, 0.147mmol).Reactant mixture is stirred under positive pressure of nitrogen power spend the night (18 hours).By reactant mixture concentrate drying and in high vacuum dry.Impure material is carried out on Biotage65M to the purified by flash chromatography (DCM with 1:1: ethyl acetate 500mL is written into, rinse, then the DCM of 1:4: ethyl acetate is until elute product (the second speckle), then gradient is pure ethyl acetate, then uses the DCM:MeOH eluting resultant product of 25:1).Merge fraction and concentrate in rotary evaporator.Residue is again concentrated and obtained 4-((5R by DCM/ hexane, 7R)-7-hydroxy-5-methyl base-6,7-dihydro-5H-cyclopenta [d] pyrimidine-4-yl) piperazine-1-carboxylic acid tert-butyl ester (mainly) and 4-((5R, 7S)-7-hydroxy-5-methyl base-6,7-dihydro-5H-cyclopenta [d] pyrimidine-4-yl) mixture (9.35g of piperazine-1-carboxylic acid tert-butyl ester (less important), 95.3% yield), it is cream-coloured foam.LC/MS(APCI+)m/z335[M+H]+。By integration carbinol methine, 1H NMR (CDCl3) shows 88%de.

step 10: by 4-nitrobenzoyl chloride (4.27g, 23.0mmol) be added to 4-((5R, 7R)-7-hydroxy-5-methyl base-6,7-dihydro-5H-cyclopenta [d] pyrimidine-4-yl) piperazine-1-carboxylic acid tert-butyl ester (7.0g, 20.9mmol) and the 0 ℃ solution of triethylamine (4.38ml, 31.4mmol) in DCM (110mL) in.Reactant mixture, in stirred overnight at room temperature, is added to saturated NaHCO afterwards ₃.Mixture is stirred 10 minutes, then extract with DCM.By dry the extract merging (Na ₂sO ₄), filter, and concentrated.Thick material is carried out on Biotage65M to the purified by flash chromatography (hexane with 3:1: ethyl acetate is written into product; then use the hexane of 2:1: eluent ethyl acetate goes out 4-((5R; 7R)-5-methyl-7-(4-nitro benzoyl oxygen base)-6,7-dihydro-5H-cyclopenta [d] pyrimidine-4-yl) piperazine-1-carboxylic acid tert-butyl ester and a small amount of mixed fraction).Then use the hexane of 1:2: eluent ethyl acetate 4-((5R, 7S)-5-methyl-7-(4-nitro benzoyl oxygen base)-6,7-dihydro-5H-cyclopenta [d] pyrimidine-4-yl) piperazine-1-carboxylic acid tert-butyl ester.The fraction that contains product is concentrated and obtains 4-((5R through rotary evaporation; 7R)-5-methyl-7-(4-nitro benzoyl oxygen base)-6; 7-dihydro-5H-cyclopenta [d] pyrimidine-4-yl) piperazine-1-carboxylic acid tert-butyl ester (8.55g; 84.5% yield), it is yellow foam.LC/MS(APCI+)m/z484[M+H]+。1H NMR (CDCl3) shows single diastereomer).The fraction that contains other diastereomer is concentrated and obtains 4-((5R through rotary evaporation; 7S)-5-methyl-7-(4-nitro benzoyl oxygen base)-6; 7-dihydro-5H-cyclopenta [d] pyrimidine-4-yl) piperazine-1-carboxylic acid tert-butyl ester (0.356g; 3.52% yield), it is brown foam.LC/MS(APCI+)m/z484[M+H]+.

step 11: by LiOH-H ₂o (0.499g; 11.9mmol) be added to 4-((5R; 7R)-5-methyl-7-(4-nitro benzoyl oxygen base)-6; 7-dihydro-5H-cyclopenta [d] pyrimidine-4-yl) piperazine-1-carboxylic acid tert-butyl ester (2.30g, 4.76mmol) is at the THF:H of 2:1 ₂in 0 ℃ of solution in O (40mL).Reactant mixture is warmed to room temperature and stirs 1 hour.THF is removed through rotary evaporation, add saturated NaHCO ₃, and mixture is extracted with ethyl acetate.By the saturated NaHCO of extract merging ₃washing (1X), dry (Na ₂sO ₄), filter, and concentrated the 4-((5R that obtains, 7R)-7-hydroxy-5-methyl base-6,7-dihydro-5H-cyclopenta [d] pyrimidine-4-yl) piperazine-1-carboxylic acid tert-butyl ester (1.59g, 100.0% yield), it is yellow foam.HPLC after post processing produces the purity of >98 area %.LC/MS(APCI+)m/z335[M+H]+。Use similar approach to prepare 4-((5R, 7S)-7-hydroxy-5-methyl base-6,7-dihydro-5H-cyclopenta [d] pyrimidine-4-yl) piperazine-1-carboxylic acid tert-butyl ester.

step 12: by 4MHCl/ diox (11.2ml, 44.9mmol) be added to 4-((5R, 7R)-7-hydroxy-5-methyl base-6,7-dihydro-5H-cyclopenta [d] pyrimidine-4-yl) piperazine-1-carboxylic acid tert-butyl ester is (in the solution in 0.600g, 1.79mmol) diox (15mL).Reactant mixture is stirred under nitrogen in room temperature spend the night (20 hours).By mixture concentrate drying dry on fine vacuum line.Thick material is suspended in ether, ultrasonic, and stir 5 minutes.Solid is filtered and separated through medium frit funnel under nitrogen pressure, use ether drip washing, dry under nitrogen pressure, and be further dried and obtain (5R on fine vacuum line, 7R)-5-methyl-4-(piperazine-1-yl)-6,7-dihydro-5H-cyclopenta [d] pyrimidin-7-ol dihydrochloride (0.440g, 79.8% yield), it is yellow powder.LC/MS(APCI+)m/z235。Use similar approach preparation (5R, 7S)-5-methyl-4-(piperazine-1-yl)-6,7-dihydro-5H-cyclopenta [d] pyrimidin-7-ol dihydrochloride.

step 13: by 2-(4-chlorphenyl) methyl acetate (36.7g, 199mmol) and paraformaldehyde (6.27g, 209mmol) be dissolved in/be suspended in DMSO (400mL) and use NaOMe (537mg, 9.94mmol) to process.Mixture, stirring at room temperature 2 hours, is analyzed and shown through the TLC of thick material.Pour reactant mixture into icy water (700mL; White milk) in and by adding 1MHCl solution neutralization.Water layer is extracted with ethyl acetate (3X), and merges organic layer.Organic layer is washed with water (2X), with salt water washing (1X), separate, through MgSO ₄dry, filter, and vacuum concentration obtains crude product, it is yellow oil.Residue is loaded on the large frit filter that contains silica gel and with the hexane of 9:1: eluent ethyl acetate is until start to collect initial substance/alkene.Then the hexane with 1:1 by filler: eluent ethyl acetate is until elute pure expection product completely.Concentrated pure fraction obtains 2-(4-chlorphenyl)-3-hydroxy methyl propionate, and it is colorless oil (39.4g, 92%).

step 14: 2-(4-chlorphenyl)-3-hydroxy methyl propionate (39.4g, 184mmol) is dissolved in DCM (500mL) and uses TEA (64.0mL, 459mmol) to process.Solution is cooled to 0 ℃ and also slowly processes with MsCl (15.6mL, 202mmol), then stir 30 minutes, analyze and shown through TLC.Solution is distributed with 1N HCl solution, and water layer is extracted once with DCM.By the organic layer merging with 1N HCl solution washing once more than, separate, use rare NaHCO ₃solution washing, and separate.By organic layer through MgSO ₄dry, filter, and vacuum concentration obtains orange.Residue is loaded into the hexane of also using 9:1 on the large frit filter with silica filler: eluent ethyl acetate, analyze and show through TLC, obtain pure expection product.Concentrated pure fraction obtains 2-(4-chlorphenyl) acrylic acid methyl ester., and it is colorless oil (30.8g, 85%).At 0 ℃, the solution of this 2-(4-chlorphenyl) acrylic acid methyl ester. (500mg, 2.54mmol) in THF (1.35mL) is added to i-PrNH ₂in the solution of (217uL, 2.54mmol) stirring in THF (5.0mL).Reactant mixture, in stirred overnight at room temperature, has been shown through lcms analysis.Boc2O (584uL, 2.54mmol) is added in the amine stirring through pipet.Reactant mixture is stirred and spent the night, shown through LCMS and the TLC analysis of mixture.Solution for vacuum concentration is obtained to 3-(tert-butoxycarbonyl (isopropyl) amino)-2-(4-chlorphenyl) methyl propionate, and it is colorless oil (854mg, 94%).LC/MS(APCI+)m/z256.1[M-Boc]+.

step 15: by 3-(tert-butoxycarbonyl (isopropyl) amino)-2-(4-chlorphenyl) methyl propionate (133g, 374mmol) be dissolved in THF (1.0L) and with KOTMS (56.0g, 392mmol) in room temperature treatment.Mixture is stirred and spent the night, shown through the lcms analysis of thick material.Mixture vacuum concentration is obtained to wet foam, its dried overnight under vacuum is obtained to 3-(tert-butoxycarbonyl (isopropyl) amino)-2-(4-chlorphenyl) potassium propionate, it is white solid (148.7g, 105%).LC/MS(APCI+)m/z242.1[M-Boc-K]+.

step 16:by 3-(tert-butoxycarbonyl (isopropyl) amino)-2-(4-chlorphenyl) potassium propionate (77.2g, 203mmol) be dissolved in THF (515mL) and with pivaloyl chloride (26.3mL, 213mmol) in room temperature treatment.Mixture is stirred 3 hours, form mixed acid anhydride.(S)-4-Bian Ji oxazolidine-2-ketone (46.1g, 260mmol) is dissolved in THF (600mL) and in separation flask and is cooled to-78 ℃.N-BuLi for solution (102mL2.50M hexane solution, 254mmol) is processed and stirred one hour.The anhydride solution of preparation is added in the Li-oxazolidone of stirring through intubate, and mixture is warmed to ambient temperature overnight.Mixture, by adding saturated ammonium chloride solution to carry out cancellation, is then distributed between more water and ethyl acetate.Water layer is extracted to several times, and merge organic layer.Organic layer is washed with water, then use salt water washing, separate, through MgSO ₄dry, filter, and vacuum concentration.By residue through chromatography (silica gel, hexane with 4:1: eluent ethyl acetate) purification/separation (diastereomer) diastereomer of obtaining separating completely, it is the grease of thickness: (R)-3-((S)-4-benzyl-2-Yang Dai oxazolidine-3-yl)-2-(4-chlorphenyl)-3-oxopropyl (isopropyl) t-butyl carbamate (12.16g, 24% sour racemic compound based on 1/2) and (S)-3-((S)-4-benzyl-2-Yang Dai oxazolidine-3-yl)-2-(4-chlorphenyl)-3-oxopropyl (isopropyl) t-butyl carbamate (39.14g, 77% sour racemic compound based on 1/2).LC/MS(APCI+)m/z401.2[M-Boc]+.

step 17: in room temperature by LiOH-H ₂o (168mg, 4.00mmol) is added in the agitating solution of THF (30mL) and water (15mL) until its dissolving.Hydrogen peroxide for mixture (the 35%wt. aqueous solution of 658uL, 8.00mmol) is processed and stirring at room temperature 10 minutes.Reactant mixture is cooled to 0 ℃ and last 10 minutes and dropwise add (S)-3-((the S)-4-benzyl-2-Yang Dai oxazolidine-3-yl) solution of-2-(4-chlorphenyl)-3-oxopropyl (isopropyl) t-butyl carbamate (1.00g, 2.00mmol) in THF (15mL) through addition funnel with ice bath.Mixture, in stirred overnight at room temperature, has been shown through the lcms analysis of thick material.Reactant mixture is cooled to 0 ℃, then lasts 10 minutes through addition funnel 1M Na ₂sO ₃(9.00mL) solution-treated.After having added, mixture is warmed to room temperature and reaches 10 minutes.Mixture is concentrated, remove THF, then dilute with water.By ethyl acetate washed twice (discarding) for water layer.Water layer is distributed by ethyl acetate, then under agitation dropwise process with 1M HCl, until reach pH2-3.Water layer is extracted with ethyl acetate to twice, and merges organic layer.By organic layer salt water washing, separate, through MgSO ₄dry, filter, and vacuum concentration.By colorless oil product high vacuum dry 1 hour, obtain (S)-3-(tert-butoxycarbonyl (isopropyl) amino)-2-(4-chlorphenyl) propanoic acid, it is grease/foam (685mg, 100%) .LC/MS (APCI+) m/z242.1[M-Boc of thickness]+.

step 18: by (5R, 7R)-5-methyl-4-(piperazine-1-yl)-6,7-dihydro-5H-cyclopenta [d] pyrimidin-7-ol dihydrochloride (2.92g, 9.51mmol) with (S)-3-(tert-butoxycarbonyl (isopropyl) amino)-2-(4-chlorphenyl) propanoic acid (3.25g, 9.51mmol) solution in DCM (40mL) and DIEA (5.0mL, 28.7mmol) was stirring at room temperature 10 minutes.HBTU (3.61g, 9.51mmol) is added in mixture.By mixture stirring at room temperature 1 hour.Except desolventizing, and residue is dissolved in ethyl acetate (500mL) and washes (6X100mL) with water.Organic facies is dried and is concentrated.Make residue carry out column chromatography purification (through EtOAc-DCM/MeOH (20:1) eluting) and obtain (S)-2-(4-chlorphenyl)-3-(4-((5R, 7R)-7-hydroxy-5-methyl base-6,7-dihydro-5H-cyclopenta [d] pyrimidine-4-yl) piperazine-1-yl)-3-oxopropyl (isopropyl) t-butyl carbamate (3.68g, 69%).LC/MS(APCI+)m/z558.2[M+H]+.

step 19: by (S)-2-(4-chlorphenyl)-3-(4-((5R, 7R)-7-hydroxy-5-methyl base-6,7-dihydro-5H-cyclopenta [d] pyrimidine-4-yl) piperazine-1-yl)-3-oxopropyl (isopropyl) t-butyl carbamate (2.50g, 4.48mmol) be dissolved in diox (22.4mL) and with the solution (22.4mL, 89.6mmol) in 4M HCl diox in room temperature treatment.The solution stirring of gained is spent the night, shown through the lcms analysis of thick material.By solution for vacuum concentration, obtain jelly, be dissolved in minimum methanol (10mL).Solution is transferred in the ether (300mL) of stirring through pipet, obtains white expection product precipitate.Walk to approximately one half when being added to, white depositions is molten into yellow jelly.Material vacuum concentration is obtained to yellow jelly, the hold over night that reduced pressure obtains (S)-2-(4-chlorphenyl)-1-(4-((5R, 7R)-7-hydroxy-5-methyl base-6,7-dihydro-5H-cyclopenta [d] pyrimidine-4-yl) piperazine-1-yl)-3-(isopropylamino) third-1-ketone dihydrochloride, it is buff powder (2.14g, 90%). ¹HNMR(D ₂O,400

8.39(s,1H),7.37-7.35(d,J=8.4Hz,2H),7.23-7.20(d,J=8.4Hz,2H),5.29-5.25(m,1H),4.33-4.29(m,1H),4.14-4.10(m,1H),3.89-3.19(m,11H),2.23-2.17(m,1H),2.08-1.99(m,1H),1.20-1.18(m,6H),0.98-0.96(d,J=6.8Hz,3H).MS(APCI+)[M+H] ⁺458。

Embodiment 2 body outer cell proliferations are measured

The external usefulness of the compound of formula I and the combination of some specific chemotherapeutant can be used

fluorecyte survival mensuration (can be by Promega Corp., Madison, WI is commercially available) measure.This homogenizing is measured the recombinant expressed (US5583024 of (homogeneous assay) method based on Homoptera luciferase; US5674713; US5700670) number (Crouch et al (1993) J.Immunol.Meth.160:81-88 that quantitatively determines living cells in culture of the ATP (indicant of metabolic activity cell) and based on existing; US6602677).Described

measure and carry out with 96 or 384 hole patterns, make it can carry out automatic high flux screening (HTS) (Cree et al (1995) AntiCancer Drugs6:398-404).Described homogenizing measurement operation comprises single agents

directly be added in the cell of hatching in the supplementary culture medium of serum.Do not need washed cell, remove culture medium and repeatedly move liquid step.Adding in reagent mixed 10 minutes, in 384-hole pattern, system detects and is low to moderate 15 cells/well.

Pattern that described homogenizing " adds-mix-measure (add-mix-measure) " causes cytolysis and produces and the proportional fluorescence signal of amount of ATP of existence.The amount of ATP is directly proportional to the cell number existing in culture.Described measure and produce " wide variety of glow-type " fluorescence signal (being produced by luciferase reaction), it has the half-life that is conventionally greater than five hours, the culture medium that depends on cell type and use.With relative luminous unit (RLU) reflection living cells.Make substrate (beetle fluorescein (Beetle Luciferin)) oxidative decarboxylation by restructuring LUC Photinus pyralis LUC Photinus pyralis FL, be accompanied by ATP is changed into AMP and produces photon.The half-life extending makes it no longer to need to use reagent syringe and makes the processing of the continuous or many plates batch mode of many plates have motility.This cell proliferating determining can be used for various porous patterns, for example 96 or 384 hole patterns.Can pass through photometer or CCD camera imaging device record data.Present luminous output, it is the relative light unit (RLU) of measuring in time.

Embodiment 3 in-vivo tumour xenograft usefulness

The usefulness of representative combination of the present invention is by implanting the allograft of cancerous cell or xenograft in rodent and tumor animal is carried out to in-vivo measurement by described combined treatment.Variable results can be depending on cell line, in tumor cell, have or do not exist some sudden change, give order, dosage regimen and the other factors of compound and be expection.Tested mice is processed with medicine or contrast (vehicle) and last some weeks or monitor for more time time, cell death logarithm value and the tumor suppression to measure tumor doubling.For the results are shown in accompanying drawing of the representative combination of the present invention who tests in this model.The bright representative combination of tables of data in accompanying drawing provides than giving separately each medicine the result of improving.

Embodiment 4 measures PTEN state

PTEN state can be measured by any proper method known in the art.In an example, use IHC.Selectively, can use western blot analysis.Antibody to PTEN is available commercially (Cell Signaling Technology, Beverly, MA, Cascade Biosciences, Winchester, MA).Be described in Neshat for the IHC of PTEN state and the exemplary operation of western blot analysis, M.S.et al.Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR, Proc.Natl Acad.Sci.USA98, 10314 – 10319 (2001) and Perren, A., et.al.Immunohistochemical Evidence of Loss of PTEN Expression in Primary Ductal Adenocarcinomas of the Breast, American Journal of Pathology, Vol.155, No.4, October1999.In addition can differentiate by technology known in the art to AKT sudden change, PI3K sudden change and the Her2/ErbB2 relevant cancer that increases.

Embodiment 5 cell survivals are measured

Cell is inoculated in to the 384-orifice plate (Catalog353962 of black, clear bottom with the density of 1500 cells/well; Becton Dickinson; Franklin Lakes, NJ) in and at 37 ℃, 5%CO ₂overnight incubation to 1.5 day.Then the serial dilution of GDC-0068, GDC-0973 or its combination be added in cell and hatch other 96 hours.According to determining that by measuring adenosine triphosphate (ATP) level in cell (test kit is measured in the survival of CellTiter-Glo fluorecyte to cell survival rate described in the operating instruction of manufacturer; CatalogG7573; Promega, Madison, WI).Fluorescence signal is recorded in the many label reader (PerkinElmer of EnVision2101; Waltham, MA) on.

Suppressing percent calculates by following: deduct the relative light unit (RLU) that is exposed to the cell that GDC-0068 and GCD-0973 combine divided by the quotient of RLU of cell that is exposed to DMSO with 1, as follows:

% inhibition=1-(RLU _combination/ RLU _dMSO)

BLISS analyzes the % of expection is suppressed to (E=E _gDC-0068+ E _gDC-0973-E _gDC-0068× E _gDC-0973) suppress E with the % of laboratory observation _oBScompare.The % that the % that BLISS scoring is expection suppresses E and laboratory observation suppresses E _oBSdifference (Δ E=E _oBS-E).

BLISS scoring is quantitative shows to be greater than simple summation action from potentiation degree and the positivity BLISS scoring of single medicine.Think that being greater than 250 overall BLISS scoring is viewed strong synergism within the scope of test concentrations.

The example of combination usefulness is shown as the thermal map of following three kinds of cancerous cell lines: A2058, has PTEN defect and B-RAF ^v600Ethe K-1735 (referring to Fig. 9) of sudden change; HCT-116, has PIK3CA ^h1047Rand KRAS ^g13Dcolorectal carcinoma (CRC) cell line (referring to Fig. 5) of sudden change; And NCI-H2122, there is KRAS ^g12Cnonsmall-cell lung cancer (NSCLC) cell line (referring to Fig. 7) of sudden change.In all three kinds of cell lines the GDC-0068 concentration between 0.37 and 10 μ M and the GDC-0973 concentration between 0.062 and 0.56 μ M observe for individually dosed to having >=the strong synergism of 15 BLISS scoring.

For whether the further synergism between research GDC-0068 and GDC-0973 depends on the activation of RAS/RAF and/or PI3K/Akt approach, in a series of cell line derived from melanoma patients, compare combined effect: MALME3M B-RAF ^v600Ethe MALME3 normal skin fibroblast of metastasis melanin tumor cell line and patient's coupling.MALME3M cell has shown the sensitivity to low concentration GDC-0973, and also observes strong synergism at the GDC-0973 of low concentration and the GDC-0068 of wide concentration range, although lack the single medicine activity (referring to Figure 28) of GDC-0068.On the contrary, MALME3 cell to GDC-0973 opposing and with the combination of GDC-0068 in do not observe synergism (referring to Figure 29).Similarly, NCI-BL2122, it is the normal B lymphoblast that derives from the patient identical with NSCLC cell line NCI-H2122, it does not demonstrate collaborative the replying (referring to Figure 30) to GDC-0973 and GDC-0068 combination yet, this and strong synergism contrary (referring to Fig. 7) observed in NCI-H2122 cell.The treatment benefit that these results show the combination of MEK and Akt inhibitor optionally therein two kinds of approach of RAS/RAF approach or PI3K/Akt and RAS/RAF be in the cancerous cell of activation and observe.

Embodiment 6 western blot analyses

At culture dish (10cm ²) in 2,000,000 cells of inoculation 10mL volume, then at 37 ℃ at 5%CO ₂overnight incubation (approximately 16 hours).Cell is exposed to 1 and 3 μ M GDC-0068,0.25 and 0.75 μ M GDC-0973 or 1 μ M GDC-0068+0.25 μ M GDC-0973 reaches 3 hours.After exposure, cold phosphate buffered saline (PBS) (PBS) for cell is washed and is being supplemented with protease inhibitor (Roche, Germany), 1mM phenyl methanesulfonamide acyl fluorides (PMSF) and from Sigma (St.Louis, MO) inhibitors of phosphatases mixture 1 and 2 from cracking in 1 × cell extraction buffer of Biosource (Carlsbad, CA).Use Bradford method (Bio-Rad protein determination (Bio-Rad; Hercules, CA) determine protein concentration.For immunoblotting, same protein amount by electrophoresis through three-glycine 4-20% gradient gel (Invitrogen; Carlsbad, CA) separately; Use from the Criterion system of Bio-Rad and operation scheme by protein delivery to NC Nitroncellulose film.

Unless otherwise mentioned, use following antibody, it is all from Cell Signaling Technologies (Beverly, MA):

Anti-pAkt (S473)

Anti-pAkt (T308)

Anti-pMEK1/2 (S217/221)

Anti-pFoxO1 (T24)/FoxO3a (T32)

Anti-pPRAS40 (T246)

Anti-p4EBP1 (T37/46)

Anti-pERK1/2 (T202/Y204)

Anti-pTSC2 (T1462)

Anti-pS6 (S235/236)

Anti-pS6 (S240/244)

The PARP of poly-(ADP-ribose) polymerase (PARP) and cracking

GAPDH is (from Advanced ImmunoChemical; Long Beach, CA)

In order to study the effect of described combination to Akt and the conduction of MEK signal, being exposed in the HCT-116CRC cell of 1 and 3 μ M GDC-0068,0.25 and 0.75 μ M GDC-0973 or 1 μ M GDC-0068 and 0.25 μ M GDC-0973 combination, evaluate the downstream targets of Akt and MEK by western blot, wherein observe synergism.As shown in Figure 24, observe in described combination the downstream targets of Akt and MEK combination knock down effect, and the enhancing of the PARP of some targets such as pTSC2, pS6 (s235/236 and S240/244), PARP and cracking knock down effect, this demonstrates than the single medicine in the more independent use of high dose even and better knocks down effect.

Embodiment 7 Flow Cytometry Assays

HCT-116 cell is inoculated in the tissue culturing plate of 96-hole.At 37 ℃, 5%CO ₂after night incubation, cell is exposed to increase in the GDC-0068 of concentration or GDC-0973 or combination reaches 4 days.In order to detect apoptosis, 100 μ L cell suspensions are added to and contain 4mMCaCl ₂, 5 μ L anchorin V-Fluorescein isothiocyanate (FITC) (BD Pharmingen; Franklin Lakes, NJ) and 100 μ L PBS of 5 μ g/mL propidium iodides (PI) in.Mixture is hatched on ice 30 minutes and by (the BD Biosciences of flow cytometer for cell; SanJose, CA) analyze.

Propidium iodide-(PI) or the percent of anchorin V-(AV) positive cell are at the combination Alignment measuring of each single medicine or GDC-0068 and GDC-0973, and the synergism of cell death induction is analyzed through BLISS.Than the single medicine of each independent use, described combination causes PI ⁺/ AV ⁺the percent of cell increases, and observes strong synergism (BLISS scoring>=15) at 0.37-10 μ M GDC-0068 and 0.185-0.556 μ M GDC-0973.Therefore, the combination of GDC-0068 and GDC-0973 also causes the synergism to the cell death induction in HCT-116 cell.

In addition,, because numerous modifications and variations will be apparent to those skilled in the art, therefore do not expect to limit the invention to accurate formation and the method for demonstration described above.Correspondingly, can think, all suitable modifications and equivalent are within the defined scope of the invention of following claims.

Claims (28)

1. Combination, described in be combined as compound or its pharmaceutical salts of formula I and be selected from GDC-0973, PD-0325901 or the combination of the medicine of their pharmaceutical salts, the compound of described formula I is:

   

   Described combination is disposed excess proliferative disease for preventative or therapeutic.
2. 2. the combination of claim 1, wherein said excess proliferative disease is cancer.
3. 3. the combination of claim 2, wherein said cancer is relevant to PTEN sudden change.
4. 4. the combination of claim 2, wherein said cancer is suddenlyd change to AKT, is crossed and express or increase relevant.
5. 5. the combination of claim 2, wherein said cancer is relevant to PI3K sudden change.
6. 6. the combination of claim 2, wherein said cancer is relevant to Her2/ErbB2 amplification.
7. 7. the combination of any one in claim 2-6, wherein cancer is selected from mesothelioma, carcinoma of endometrium, cancer of pancreas, breast carcinoma, pulmonary carcinoma, ovarian cancer, carcinoma of prostate, melanoma, gastric cancer, colon cancer, renal carcinoma, head and neck cancer and glioma.
8. 8. the combination of any one in claim 1-7, the wherein combination of the compound of giving construction I or its pharmaceutical salts and GDC-0973.
9. 9. the combination of any one in claim 1-7, the wherein combination of the compound of giving construction I or its pharmaceutical salts and PD-0325901.
10. 10. the combination of any one in claim 1-9, wherein compound or its salt and described one or more medicines of while giving construction I.
11. The combination of any one in 11. claim 1-9, wherein compound or its salt and described one or more medicines of priority giving construction I.
12. The combination of any one in 12. claim 1-9, wherein starts to give described one or more medicines for approximately 1 to approximately 10 day before giving described combination.
13. The combination of any one in 13. claim 1-9, wherein compound or its salt of approximately 1 to approximately 10 day beginning giving construction I before giving described combination.
14. The combination of any one in 14. claim 1-9, is wherein starting on the same day compound or its salt of giving construction I and is giving described one or more medicines.
15. The combination of any one in 15. claim 1-7, the wherein combination of the compound of giving construction I or its pharmaceutical salts and GDC-0973, and described cancer is cancer of pancreas.
16. The combination of any one in 16. claim 1-7, the wherein combination of the compound of giving construction I or its pharmaceutical salts and GDC-0973, and described cancer is nonsmall-cell lung cancer.
17. The combination of any one in 17. claim 1-7, the wherein combination of the compound of giving construction I or its pharmaceutical salts and GDC-0973, and described cancer is breast carcinoma.
18. The combination of any one in 18. claim 1-7, the wherein combination of the compound of giving construction I or its pharmaceutical salts and GDC-0973, and described cancer is colon cancer.
19. The combination of any one in 19. claim 1-7, the wherein combination of the compound of giving construction I or its pharmaceutical salts and GDC-0973, and described cancer is melanoma.
20. The compound of 20. formula I or its pharmaceutical salts, for improving the therapeutic use for the patient's of excess proliferative disease treatment quality of life together with being selected from the medicine of GDC-0973, PD-0325901 or its pharmaceutical salts.
21. 21. combinations, it is compound or its pharmaceutical salts of a) formula I; And b) being selected from the combination of one or more medicines of GDC-0973, PD-0325901 or its pharmaceutical salts, described combination is used for the treatment of excess proliferative disease.
22. 22. combinations, it is compound or its pharmaceutical salts of a) formula I; And b) being selected from the combination of one or more medicines of GDC-0973, PD-0325901 or its pharmaceutical salts, described combination is used for the treatment of the disease or the disease that are regulated by AKT kinases.
23. Being combined in for the preparation of the purposes in the medicine of excess proliferative disease in treatment mammal of the compound of 23. formula I or its pharmaceutical salts and GDC-0973, PD-0325901 or its pharmaceutical salts.
24. Being combined in for the preparation of the purposes in the medicine of the disease being regulated by AKT kinases in treatment mammal or disease of the compound of 24. formula I or its pharmaceutical salts and GDC-0973, PD-0325901 or its pharmaceutical salts.
25. 25. test kits, comprise compound or its pharmaceutical salts, container and package insert or the label of formula I, described package insert or label indicate the compound and one or more medicines that are selected from GDC-0973, PD-0325901 or its pharmaceutical salts of the formula I that is used for the treatment of excess proliferative disease.
26. 26. products, comprise compound or its pharmaceutical salts with formula I and one or more medicines that are selected from GDC-0973, PD-0325901 or its pharmaceutical salts; Described product is as the compound artifact for separately, simultaneously or successively using at overmedication proliferative disease.
27. 27. are used for the treatment of the method for excess proliferative disease in mammal, comprise to described mammal and combining, the described combination that is combined as compound or its pharmaceutical salts of formula I and is selected from the other medicine of GDC-0973, PD-0325901 or its pharmaceutical salts, the compound of described formula I is:

    
28. 28. are used for the treatment of the disease that regulated by AKT kinases in mammal or the method for disease, comprise to described mammal and give a) compound or its pharmaceutical salts of formula I; And b) be selected from one or more medicines of GDC-0973, PD-0325901 or its pharmaceutical salts.

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